IGF-1R ANTIBODY-DRUG-CONJUGATE
AND ITS USE FOR THE TREATMENT OF CANCER
The present invention relates to an antibody-drug-conjugate capable of binding
to the IGF-1R. From one aspect, the invention relates to an antibody-drug-conjugate
comprising an antibody capable of binding to IGF-1R, said antibody being conjugated
to at least one drug selected from derivatives of dolastatin 10 and auristatins. The
invention also comprises method of treatment and the use of said antibody-drugconjugate
for the treatment of cancer.
Background of the invention
The insulin- like growth factor 1 receptor called IGF-1R (or sometimes IGF1R or
IGF-IR) is a receptor with tyrosine kinase activity having 70% homology with the
insulin receptor IR. IGF-IR is a glycoprotein of molecular weight approximately
350,000. It is a hetero-tetrameric receptor of which each half -linked by disulfide
bridges- is composed of an extracellular a-subunit and of a transmembrane b-subunit.
IGF-IR binds IGF1 and IGF2 with a very high affinity (Kd # 1 nM) but is equally
capable of binding to insulin with an affinity 100 to 1000 times lower. Conversely, the
IR binds insulin with a very high affinity although the IGFs only bind to the insulin
receptor with a 100 times lower affinity. The tyrosine kinase domain of IGF-IR and of
IR has a very high sequence homology although the zones of weaker homology
respectively concern the cysteine-rich region situated on the a-subunit and the Cterminal
part of the b-subunit. The sequence differences observed in the a-subunit are
situated in the binding zone of the ligands and are therefore at the origin of the relative
affinities of IGF-IR and of IR for the IGFs and insulin respectively. The differences in
the C-terminal part of the b-subunit result in a divergence in the signalling pathways of
the two receptors; IGF-IR mediating mitogenic, differentiation and antiapoptosis
effects, while the activation of the IR principally involves effects at the level of the
metabolic pathways.
The cytoplasmic tyrosine kinase proteins are activated by the binding of the
ligand to the extracellular domain of the receptor. The activation of the kinases in turn
involves the stimulation of different intra-cellular substrates, including IRS-1, IRS-2,
She and Grb 10. The two major substrates of IGF-IR are IRS and She which mediate,
by the activation of numerous effectors downstream, the majority of growth and
differentiation effects connected with the attachment of the IGFs to this receptor. The
availability of substrates can consequently dictate the final biological effect connected
with the activation of the IGF-IR. When IRS-1 predominates, the cells tend to
proliferate and to transform. When She dominates, the cells tend to differentiate. It
seems that the route principally involved for the effects of protection against apoptosis
is the phosphatidyl-inositol 3-kinases (PI 3-kinases) route.
The role of the IGF system in carcinogenesis has become the subject of intensive
research in the last ten years. This interest followed the discovery of the fact that in
addition to its mitogenic and antiapoptosis properties, IGF-IR seems to be required for
the establishment and the maintenance of a transformed phenotype. In fact, it has been
well established that an overexpression or a constitutive activation of IGF-IR leads, in a
great variety of cells, to a growth of the cells independent of the support in media
devoid of foetal calf serum, and to the formation of tumors in nude mice. This in itself is
not a unique property since a great variety of products of overexpressed genes can
transform cells, including a good number of receptors of growth factors. However, the
crucial discovery which has clearly demonstrated the major role played by IGF-IR in
the transformation has been the demonstration that the IGR-1R cells, in which the gene
coding for IGF-IR has been inactivated, are totally refractory to transformation by
different agents which are usually capable of transforming cells, such as the E5 protein
of bovine papilloma virus, an overexpression of EGFR or PDGFR, the T antigen of
SV40, activated ras or the combination of these two last factors.
IGF-IR is expressed in a great variety of tumors and of tumor lines and the IGFs
amplify the tumor growth via their attachment to IGF-IR. Other arguments in favor of
the role of IGF-IR in carcinogenesis come from studies using murine monoclonal
antibodies directed against the receptor or using negative dominants of IGF-IR.
Actually, murine monoclonal antibodies directed against IGF-IR inhibit the
proliferation of numerous cell lines in culture and the growth of tumor cells in vivo. It
has likewise been shown that a negative dominant of IGF-IR is capable of inhibiting
tumor proliferation.
A large number of projects have been initiated to develop naked IGF-IR
antibodies for the treatment of cancers. Nevertheless, at this date, none of these projects
have been successful and there are no anti-IGF-lR antibodies on the market.
Moreover, a series of clinical trials involving anti-IGF-lR antibodies combined
to anti-EGFR antibodies in order to target both EGFR and IGF-IR, have failed as none
of these antibodies were able to treat KRAS mutant patients.
As a consequence, IGF-IR is not considered now as a major target and, in the
research of potential therapeutic antibodies, IGF-IR is no more considered as of
particular interest.
Nevertheless, it must also be noticed that endeavours to generate IGF-IR
antibodies were focussed on naked antibodies, i.e. antibodies useful by their intrinsic
properties. In this sense, IGF-IR is considered as a target not suitable for the generation
of an ADC such as an antibody-drug-conjugate (referred as "ADC") as IGF-IR is
described as a target also widely expressed by normal cells, including blood vessels. In
this sense, it can be noticed that the most recent IGF-IR antibody, i.e. AVE1642, is
developed as a naked antibody not armed with a drug. It is the same with the other IGFIR
antibodies currently in development and with all those which failed in clinical trials.
In this context, the invention relates to an ADC or conjugate and its use for the
treatment of cancer, and more particularly IGF-lR-expressing cancers.
ADCs combine the binding specificity of an antibody with the potency of drugs
such as, for example, cytotoxic agents. The technology associated with the development
of monoclonal antibodies, the use of more effective drugs and the design of chemical
linkers to covalently bind these components, has progressed rapidly in recent years .
The use of ADCs allows the local delivery of drugs which, if administered as
unconjugated drugs, may result in unacceptable levels of toxicity to normal cells.
In other words, maximal efficacy with minimal toxicity is sought thereby.
Efforts to design and refine ADC have focused on the selectivity of antibody as well as
drug mechanism of action, drug-linking, drug/antibody ratio (loading or DAR), and
drug-releasing properties . Drug moieties may impart their cytotoxic and cytostatic
effects by mechanisms including tubulin binding, DNA binding, proteasome,
impairement of ribosome function, protein synthesis and/or topoisomerase inhibition.
Some cytotoxic drugs tend to be inactive or less active when conjugated to large
antibody.
Each antibody must be characterized separately, an appropriate linker designed,
and a suitable cytotoxic agent identified that retains its potency upon delivery to tumor
cells. One must consider the antigen density on the cancer target and whether normal
tissues express the target antigen. Other considerations include whether the entire ADC
is internalized upon binding the target; whether a cytostatic or cytotoxic drug is
preferable when considering possible normal tissue exposure and/or the type and stage
of the cancer being treated; and, whether the linker connecting the antibody to the drug
payload is a cleavable or a non-cleavable linkage. Furthermore, the antibody to drug
moiety conjugation ratio must be sufficient without compromising the binding activity
of the antibody and/or the potency of the drug and without modifying physicochemical
properties of the ADC resulting on aggregation or deleterious properties regarding to
the future development process of the compound.
An ADC is a complex biological molecule and the challenges to develop an
effective ADC remain a significant issue.
Summary of the invention
The present invention intends to address this issue and relates to an ADC of the
following formula (I):
Ab-(L-D)
(I)
or a pharmaceutically acceptable salt thereof,
wherein
Ab is an antibody, or an antigen binding fragment thereof, capable of binding to
the human IGF-1R selected from:
i) an antibody which comprises the three heavy chain CDRs of sequence SEQ ID No. 1,
2 and 3 and the three light chain CDRs of sequence SEQ ID No. 4, 5 and 6;
ii) an antibody that competes for binding to IGF-1R with the antibody of i); and
iii) an antibody that binds to the same epitope of IGF-1R as the antibody of i);
L is a linker;
D is a drug moiety of the following formula (II):
wherein:
R2 is COOH, COOCH3 or thiazolyl;
R3 is H or (Ci-C )alkyl;
R is H or (Ci-C )alkyl;
m is an integer comprised between 1 and 8;
the wavy line indicates the point of attachment to L ; and
n is 1 to 12.
An embodiment of the invention relates to an ADC wherein Ab is selected from:
a) an antibody comprising the three heavy chain CDRs of sequence SEQ ID No. 7, 2
and 3 and the three light chain CDRs of sequence SEQ ID No. 9, 5 and 11;
b) an antibody comprising the three heavy chain CDRs of sequence SEQ ID No. 7, 2
and 3 and the three light chain CDRs of sequence SEQ ID No. 10, 5 and 11;
c) an antibody comprising the three heavy chain CDRs of sequence SEQ ID No. 7, 2
and 3 and the three light chain CDRs of sequence SEQ ID No. 9, 5 and 12; and
d) an antibody comprising the three heavy chain CDRs of sequence SEQ ID No. 8, 2
and 3 and the three light chain CDRs of sequence SEQ ID No. 9, 5 and 11.
An embodiment of the invention relates to an ADC wherein Ab is selected from:
a) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 13
and the three light chain CDRs of sequence SEQ ID No. 9, 5 and 11;
b) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 14
and the three light chain CDRs of sequence SEQ ID No. 10, 5 and 11;
c) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 15
and the three light chain CDRs of sequence SEQ ID No. 9, 5 and 12;
d) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 16
and the three light chain CDRs of sequence SEQ ID No. 9, 5 and 11; and
e) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 1
and the three light chain CDRs of sequence SEQ ID No. 9, 5 and 12.
An embodiment of the invention relates to an ADC wherein Ab is selected from:
a) an antibody comprising a light chain variable domain of sequence SEQ ID No. 18
and the three heavy chain CDRs of sequence SEQ ID No. 7, 2 and 3;
b) an antibody comprising a light chain variable domain of sequence SEQ ID No. 19
and the three heavy chain CDRs of sequence SEQ ID No. 7, 2 and 3;
c) an antibody comprising a light chain variable domain of sequence SEQ ID No. 20
and the three heavy chain CDRs of sequence SEQ ID No. 7, 2 and 3;
d) an antibody comprising a light chain variable domain of sequence SEQ ID No. 2 1
and the three heavy chain CDRs of sequence SEQ ID No. 8, 2 and 3; and
e) an antibody comprising a light chain variable domain of sequence SEQ ID No. 22
and the three heavy chain CDRs of sequence SEQ ID No. 7, 2 and 3.
In an embodiment the invention relates to an ADC wherein Ab is selected from:
i) the antibodies 208F2, 212A1 1, 214F8, 219D6 and 213B10;
ii) the antibodies which compete for binding to IGF-1R with the antibodies of i); and
iii) the antibodies which bind to the same epitope of IGF-1R as the antibodies of i).
An embodiment of the invention relates to an ADC wherein Ab is a humanized
antibody.
An embodiment of the invention relates to an ADC wherein Ab is selected from
an antibody comprising:
a) a heavy chain having CDR-Hl, CDR-H2 and CDR-H3 of sequences SEQ ID Nos. 7,
2 and 3, respectively, and FRl, FR2 and FR3 derived from the human germline IGHV1-
46*01 (SEQ ID No. 46), and the FR4 derived from the human germline IGHJ4*01
(SEQ ID No. 48); and
b) a light chain having CDR-L1, CDR-L2 and CDR-L3 of sequences SEQ ID Nos. 9, 5
and 11, respectively, and FR1, FR2 and FR3 derived from the human germline IGKV1-
39*01 (SEQ ID No. 47), and the FR4 derived from the human germline IGKJ4*01
(SEQ ID No. 49).
In an embodiment of the invention, Ab is selected from:
a) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 33
or any sequence exhibiting at least 80% identity with SEQ ID No. 33 and the three light
chain CDRs of sequences SEQ ID Nos. 9, 5 and 11; and
b) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 34
or any sequence exhibiting at least 80% identity with SEQ ID No. 34 and the three light
chain CDRs of sequences SEQ ID Nos. 9, 5 and 11.
In an embodiment of the invention, Ab is selected from:
a) an antibody comprising a light chain variable domain of sequence SEQ ID No. 35 or
any sequence exhibiting at least 80% identity with SEQ ID No. 35 and the three heavy
chain CDRs of sequences SEQ ID Nos. 7, 2 and 3; and
b) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 36
or any sequence exhibiting at least 80% identity with SEQ ID No. 36 and the three
heavy chain CDRs of sequences SEQ ID Nos. 7, 2 and 3.
In an embodiment of the invention, Ab is selected from:
a) an antibody comprising or conssting of a heavy chain of sequence SEQ ID No. 37 or
any sequence exhibiting at least 80% identity with SEQ ID No. 37 and a light chain of
sequence SEQ ID No. 39 or any sequence exhibiting at least 80%> identity with SEQ ID
No. 39; and
b) an antibody comprising or conssting of a heavy chain of sequence SEQ ID No. 38 or
any sequence exhibiting at least 80% identity with SEQ ID No. 38 and a light chain of
sequence SEQ ID No. 40 or any sequence exhibiting at least 80%> identity with SEQ ID
No. 40.
In an embodiment of the invention, Ab is selected from:
a) an antibody comprising a heavy chain variable domain of sequence selected
from SEQ ID Nos. 56, 62, 64, 66, 68, 70, 72, 74, 76, 78 and 80 or any sequence with at
least 80% identity with SEQ ID No.56, 62, 64, 66, 68, 70, 72, 74, 76, 78 or80; and the
three light chain CDRs of sequences SEQ ID Nos. 9, 5 and 11;
b) an antibody comprising a light chain variable domain of sequence selected
from SEQ ID Nos. 57 and 60 or any sequence with at least 80% identity with SEQ ID
Nos. 57 or 60; and the three heavy chain CDRs of sequences SEQ ID Nos. 7, 2 and 3;
and
c) an antibody comprising a heavy chain variable domain of sequence selected
from SEQ ID Nos. 56, 62, 64, 66, 68, 70, 72, 74, 76, 78 and 80 or any sequence with at
least 80% identity with SEQ ID Nos.56, 62, 64, 66, 68, 70, 72, 74, 76, 78 or 80; and a
light chain variable domain of sequence selected from SEQ ID Nos. 57 or 60 or any
sequence with at least 80%> identity with SEQ ID Nos. 57 or 60.
In an embodiment of the invention, Ab is selected from:
a) a heavy chain of sequence selected from SEQ ID Nos. 58, 63, 65, 67, 69, 71,
73, 75, 77, 79 and 8 1 or any sequence with at least 80%> identity with SEQ ID Nos. 58,
63, 65, 67, 69, 71, 73, 75, 77, 79 or 81; and
b) a light chain of sequence selected from SEQ ID Nos. 59 and 6 1 or any
sequence with at least 80%> identity with SEQ ID Nos. 59 or 6 1.
In an embodiment of the invention relates to an ADC wherein L is a linker of the
following formula (III):
(III)
wherein
L2 is (C4 -Cio)cycloalkyl-carbonyl, (C2-C )alkyl or (C2-C )alkyl-carbonyl;
W is an amino acid unit; w is an integer comprised between 0 and 5;
Y is PAB-carbonyl with PAB being y is 0 or 1;
the asterisk indicates the point of attachment to D; and
the wavy line indicates the point of attachment to Ab.
An embodiment of the invention relates to an ADC wherein L2 is of the
following formula:
wherein
the asterisk indicates the point of attachment to (W)w; and
the wavy line indicates the point of attachment to the nitrogen atom of the
maleimide moiety of formula:
embodiment of the invention, w = 0, or w = 2 and then (W)w is selected
from:
and
wherein
the asterisk indicates the point of attachment to (Y)y; and
the wavy line indicates the point of attachment to L2.
An embodiment of the invention relates to an ADC wherein L is selected from:
wherein the asterisk indicates the point of attachment to D, and the wavy line
indicates the point of attachment to Ab.
An embodiment of the invention relates to an ADC wherein (L-D) is selected
from:

(F-63)
wherein the wavy line indicates the point of attachment to Ab.
An embodiment of the invention relates to an ADC having the formula selected
from:
ĨAb-G-12)
ĨAb-G-13)
ĨAb-F-61)
(Ab-F-63)
and the pharmaceutically acceptable salts thereof,
wherein Ab is selected in the group consisting of:
i) the antibodies 208F2, 212A1 1, 214F8, 219D6 and 213B10;
ii) the antibodies which compete for binding to IGF-IR with the antibodies of i); and
iii) the antibodies which bind to the same epitope of IGF-IR as the antibodies of i).
An embodiment of the invention relates to an ADC wherein n is 2.
An embodiment of the invention relates to an ADC wherein n is 4.
An embodiment of the invention relates to an ADC for use as a medicament.
An embodiment of the invention relates to a composition comprising an ADC as
above described.
An embodiment of the invention relates to a composition further comprising a
pharmaceutically acceptable vehicle.
An embodiment of the invention relates to a composition for use in the treatment
of an IGF-lR-expressing cancer, or IGF-1R related cancers.
IGF-lR-expressing cancer or IGF-1R related cancers include tumoral cells
expressing or over-expressing whole or part of the IGF-1R at their surface.
An embodiment of the invention relates to a composition, wherein said IGF-lRexpressing
cancer is a cancer chosen from breast, colon, esophageal carcinoma,
hepatocellular, gastric, glioma, lung, melanoma, osteosarcoma, ovarian, prostate,
rhabdomyosarcoma, renal, thyroid, uterine endometrial cancer, mesothelioma, oral
squamous carcinoma and any drug resistant cancer.
An embodiment of the invention relates to a method for the treatment of an IGFlR-
expressing cancer in a subject in need thereof, comprising administering to the
subject an effective amount of at least one antibody-drug-conjugate or of a composition
according to the invention.
An embodiment of the invention relates to a kit comprising at least i) an
antibody-drug-conjugate and/or a composition as above described and ii) a syringe or
vial or ampoule in which the said antibody-drug-conjugate and/or composition is
disposed.
Detailed description of the invention
I - The Antibody (Ab)
The terms "antibody", "antibodies" "ab", "Ab", "MAb" or "immunoglobulin"
are used interchangeably in the broadest sense and include monoclonal antibodies,
isolated, engineered or recombinant antibodies (e.g., full length or intact monoclonal
antibodies), polyclonal antibodies, multivalent antibodies or multispecific antibodies
(e.g., bispecific antibodies) and also antibody fragment thereof, so long as they exhibit
the desired biological activity.
In an embodiment, the antibody of the ADC of the invention consists of a
recombinant antibody. The term "recombinant antibody" refers to an antibody that
results from the expression of recombinant DNA within living cells. A recombinant
antibody of ADC of the invention is obtained by using laboratory methods of genetic
recombination, well known by a person skilled in the art, creating DNA sequences that
would not be found in biological organisms.
In another embodiment, the antibody of the ADC of the invention consists of a
chemically synthesized antibody.
More particularly, such a molecule consists of a glycoprotein comprising at least
two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each
heavy chain comprises a heavy chain variable region (or domain) (abbreviated herein as
HCVR or VH) and a heavy chain constant region. The heavy chain constant region
comprises three domains, CHI, CH2 and CH3. Each light chain comprises a light chain
variable region (abbreviated herein as LCVR or VL) and a light chain constant region.
The light chain constant region comprises one domain, CL. The VH and VL regions can
be further subdivided into regions of hypervariability, termed complementarity
determining regions (CDR), interspersed with regions that are more conserved, termed
framework regions (FR). Each VH and VL is composed of three CDRs and four FRs,
arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1,
FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains
contain a binding domain that interacts with an antigen. The constant regions of the
antibodies may mediate the binding of the immunoglobulin to host tissues or factors,
including various cells of the immune system (e.g. effector cells) and the first
component (Clq) of the classical complement system.
By "antigen binding fragment" or "IGF-IR binding fragment" of an antibody of
the ADC according to the invention, it is intended to indicate any peptide, polypeptide,
or protein retaining the ability to bind to the target (also generally referred as antigen) of
the antibody..
In an embodiment, such "antigen binding fragments" are selected in the group
consisting of Fv, scFv (sc for single chain), Fab, F(ab') 2, Fab', scFv-Fc fragments or
diabodies, or any fragment of which the half-life time would have been increased by
chemical modification, such as the addition of poly(alkylene) glycol such as
poly(ethylene) glycol ("PEGylation") (pegylated fragments called Fv-PEG, scFv-PEG,
Fab-PEG, F(ab') 2-PEG or Fab'-PEG) ("PEG" for Poly(Ethylene) Glycol), or by
incorporation in a liposome, said fragments having at least one of the characteristic
CDRs of the antibody according to the invention. Preferably, said "antigen binding
fragments" will be constituted or will comprise a partial sequence of the heavy or light
variable chain of the antibody from which they are derived, said partial sequence being
sufficient to retain the same specificity of binding as the antibody from which it is
descended and a sufficient affinity, preferably at least equal to 1/100, in a more
preferred manner to at least 1/10, of the affinity of the antibody from which it is
descended, with respect to the target. More preferably, said "antigen binding fragments"
will be constituted of or will comprise at least the three CDRs CDR-Hl, CDR-H2 and
CDR-H3 of the heavy variable chain and the three CDRs CDR-L1, CDR-L2 and CDRL3
of the light variable chain of the antibody from which they are derived.
By "binding", "binds", or the like, it is intended that the antibody, or any antigen
binding fragment thereof, forms a complex with an antigen that is relatively stable
under physiologic conditions. Specific binding can be characterized by an equilibrium
dissociation constant of at least about lxlO 6 M. Methods for determining whether two
molecules bind are well known in the art and include, for example, equilibrium dialysis,
surface plasmon resonance, radio labelled assays and the like. For the avoidance of
doubt, it does not mean that the said antibody could not bind or interfere, at a low level,
to another antigen. Nevertheless, as an embodiment, the said antibody binds only to the
said antigen.
As used in the present specification, the expression "IGF-1R antibody" should
be interpreted as similar to "anti-IGF-lR antibody" and means an antibody capable of
binding to IGF- 1R.
In an embodiment of the present application, the epitope of the antibody is
preferentially localized into the extracellular domain of the human IGF-1R (also
referred as IGF-1R ECD).
In a particular embodiment, the antibody, or any antigen binding fragment
thereof, is capable of binding to IGF-1R with an EC50 comprised between lOxlO 10 to
lxlO 10 , and more preferentially between 8x1 0 10 to 2x1 0 10 .
The term half maximal effective concentration (EC 50) corresponds to the
concentration of a drug, antibody or toxicant which induces a response halfway between
the baseline and maximum after some specified exposure time. It is commonly used as a
measure of drug's potency. The EC50 of a graded dose response curve therefore
represents the concentration of a compound where 50% of its maximal effect is
observed. The EC50 of a quantal dose response curve represents the concentration of a
compound where 50% of the population exhibits a response, after specified exposure
duration. Concentration measures typically follow a sigmoidal curve, increasing rapidly
over a relatively small change in concentration. This can be determined mathematically
by derivation of the best-fit line.
As a preferred embodiment, the EC50, determined in the present invention,
characterizes the potency o antibody to bind o the IGF-1 R ECD exposed o human
tumor ceils. The EC50 parameter is determined using FACS analysis. The EC50
parameter reflects the antibody concentration for which 50% of the maximal binding on
the human IGF-1 R expressed o h an tumor cells is obtained. Each EC50 value was
calculated as the midpoint of the dose response curve using a four-parameter regression
curve fitting program (Prism Software). This parameter has been selected as to be
representative of physiological/pathological conditions.
The term "epitope" is a region of an antigen that is bound by an antibody.
Epitopes may be defined as structural or functional. Functional epitopes are generally a
subset of the structural epitopes and have those residues that directly contribute to the
affinity of the interaction. Epitopes may also be conformational, that is, composed of
non-linear amino acids. In certain embodiments, epitopes may include determinants that
are chemically active surface groupings of molecules such as amino acids, sugar side
chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have
specific three-dimensional structural characteristics, and/or specific charge
characteristics.
The competition for binding to IGF-1R can be determined by any methods or
techniques known by the person skilled in the art such as, without limitation,
radioactivity, Biacore, ELISA, Flow cytometry, etc. As "which competes for binding to
IGF-1R" it is meant a competition of at least 20%, preferentially at least 50% and more
preferentially at least 70%.
The determination of the binding to the same epitope can be determined by any
methods or techniques known by the person skilled in the art such as, without
limitation, radioactivity, Biacore, ELISA, Flow cytometry, etc. As "which bind to the
same epitope of IGF-1R, it is meant a competition of at least 20%, preferentially at least
50% and more preferentially at least 70%.
As above mentioned, and contrary to the general knowledge, the present
invention focuses on specific IGF-1R antibodies presenting a high ability to be
internalized following IGF-1R binding. As used herein, an antibody that "is
internalized" or that "internalized" (the two expressions being similar) is one that is
taken up by (meaning it "enters") the cell upon binding to IGF-1R on a mammalian cell.
Such an antibody is interesting as part of the ADC, so it addresses or directs the linked
cytotoxic into the targeted cancer cells. Once internalized the cytotoxic triggers cancer
cell death.
Surprisingly, the antibodies according to the invention are all presenting the
same sequences for the CDR-H2, CDR-H3 and CDR-L2, the other 3 CDRs being
different. This observation seems coherent as it is part of the general knowledge that,
regarding the binding specificity of an antibody, the CDR-H3 is described as being the
most important and the most implicated with the recognition of the epitope.
Important keys to success with ADC therapy are thought to be the target antigen
specificity and the internalization of the antigen-antibody complexes into the cancer
cells. Obviously non-internalizing antigens are less effective than internalizing antigens
to delivers cytotoxic agents. Internalization processes are variable across antigens and
depend on multiple parameters that can be influenced by antibodies.
In the ADC, the cytotoxic confers the cytotoxic activity and the used antibody is
responsible for the specificity against cancer cells, as well as a vector for entering
within the cells to correctly address the cytotoxic. Thus to improve the ADC, the
antibody can exhibit high ability to internalize into the targeted cancer cells. The
efficiency of the antibody mediated internalisation differs significantly depending on the
epitope targeted. Selection of potent internalizing IGF-1R antibodies requires various
experimental data studying not only IGF-1R downregulation but also following IGF-1R
antibody internalization into the cells.
In an embodiment, the internalization of the antibody of the ADC according to
the invention can be evaluated by immunofluorescence or FACS (Flow Cytometry) (as
exemplified hereinafter in the present application) or any method or process known by
the person skilled in the art specific for the internalization mechanism. In a preferred
embodiment, the antibody od the ADC according to the invention can induce
internalization after binding to IGF-1R of at least 30%, preferentially 50% and more
preferentially 80%.
The complex IGF-lR/antibody is internalized after binding of the antibody to the
ECD of said IGF-1R, and a reduction in the quantity of IGF-1R at the surface of the
cells is induced. This reduction can be quantified by any method known by the person
skilled in the art such as non limitative examples western-blot, FACS, and
immunofluorescence.
In one embodiment, this reduction, thus reflecting the internalization, can be
preferably measured by FACS and expressed as the difference or delta between the
Mean Fluorescence Intensity (MFI) measured at 4°C with the MFI measured at 37°C
after 4 hours incubation with the antibody.
As non limitative example, this delta is determined based on MFIs obtained with
untreated cells and cells treated with the antibody using i) breast cancer cells MCF7
after a 4 hour incubation period with the antibody herein described and ii) a secondary
antibody labelled with Alexa488. This parameter is defined as calculated with the
following formula: A(MFI4°c- MFl37°c).
This difference between MFIs reflects the IGF-1R downregulation as MFIs are
proportional to IGF-1R expressed on the cell-surface.
In an advantageous aspect, the antibodies consist of antibodies triggering a
A(MFI4°c- MFI37°c ) on MCF-7 of at least 280, preferably of at least 400.
In more details, the above mentioned delta can be measured according to the
following process, which must be considered as an illustrative and non limitative
example:
a) Treating and incubating tumor cells of interest with the antibody of
the invention in either cold (4°C) or warm (37°C) complete culture
medium;
b) Treating the treated cells of step a) and, in parallel, untreated cells
with a secondary antibody;
c) Measuring the MFI (representative of the quantity of IGF-1R present
at the surface) for the treated and the non treated cells with a
secondary labeled antibody capable of binding to the antibody of the
invention; and
d) Calculating the delta as the subtraction of the MFI obtained with the
treated cells from the MFI obtained with the non treated cells.
From this delta MFI, an internalization percentage can be determined as:
100x(MFI 4°c-MFI37°c) / MFI 4°c.
The antibodies of the ADC according to the invention, present, preferably, on
MCF7 an internalization percentage comprised between 50% and 99%, 70% and 90%,
preferentially between 75% and 87%.
A particular advantage of the antibodies herein described relies on their rate of
internalization.
It is generally known that, for an ADC, it is desirable that the used antibodies
exhibit a rapid rate of internalization, preferably within 24 hours from administration of
the antibody and, more preferably within 12 hours and, even more preferably within 6
hours.
In the present invention, the internalization rate, also referred as cell surface
bound antibody decrease or cell surface antibody decay, is expressed as tl/2 (half life)
and corresponds as the time necessary to obtain a decrease of 50% of the AMFI (this
aspect will be clearly understood regarding the following examples).
A particular advantage is that the antibodies of the ADC of the invention have a
tl/2 comprised between 5 and 25 minutes, and preferentially between 10 and 20
minutes.
A particular embodiment of the invention relates to an ADC wherein the
antibody Ab comprises three heavy chain CDRs with CDR-H2 of sequence SEQ ID No.
2 and CDR-H3 of sequence SEQ ID No. 3, and three light chain CDRs with CDR-L2 of
sequence SEQ ID No. 5.
A particular embodiment of the invention relates to an ADC wherein the
antibody Ab comprises the three heavy chain CDRs of sequences SEQ ID Nos. 1, 2 and
3 and the three light chain CDRs of sequences SEQ ID Nos. 4, 5 and 6.
An embodiment of the ADC comprises an antibody comprising the three heavy
chain CDRs comprising or consisting of the sequences SEQ ID Nos. 1, 2 and 3, or any
sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with
SEQ ID Nos. 1, 2 or3; and the three light chain CDRs comprising or consisting of the
sequences SEQ ID Nos. 4, 5 and 6, or any sequence exhibiting at least 80%, preferably
85%, 90%, 95% and 98% identity with SEQ ID Nos. 4, 5 or 6.
In another embodiment, the antibody, or any antigen binding fragment thereof,
comprises the three heavy chain CDRs comprising the sequences SEQ ID Nos. 1, 2 and
3; and the three light chain CDRs comprising the sequences SEQ ID Nos. 4, 5 and 6.
The IMGT unique numbering has been defined to compare the variable domains
whatever the antigen receptor, the chain type, or the species [Lefranc M.-P.,
Immunology Today 18, 509 (1997) / Lefranc M.-P., The Immunologist, 7, 132-136
(1999) / Lefranc, M.-P., Pommie, C , Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L.,
Thouvenin-Contet, V. and Lefranc, Dev. Comp. Immunol, 27, 55-77 (2003)]. In the
IMGT unique numbering, the conserved amino acids always have the same position, for
instance cystein 23 (lst-CYS), tryptophan 4 1 (CONSERVED-TRP), hydrophobic
amino acid 89, cystein 104 (2nd-CYS), phenylalanine or tryptophan 118 (J-PHE or JTRP).
The IMGT unique numbering provides a standardized delimitation of the
framework regions (FR1-IMGT: positions 1 to 26, FR2-IMGT: 39 to 55, FR3-IMGT:
66 to 104 and FR4-IMGT: 118 to 128) and of the complementarity determining regions:
CDR1-IMGT: 27 to 38, CDR2-IMGT: 56 to 65 and CDR3-IMGT: 105 to 117. As gaps
represent unoccupied positions, the CDR-IMGT lengths (shown between brackets and
separated by dots, e.g. [8.8.13]) become crucial information. The IMGT unique
numbering is used in 2D graphical representations, designated as IMGT Colliers de
Perles [Ruiz, M. and Lefranc, M.-P., Immunogenetics, 53, 857-883 (2002) / Kaas, Q.
and Lefranc, M.-P., Current Bioinformatics, 2, 21-30 (2007)], and in 3D structures in
IMGT/3Dstructure-DB [Kaas, Q., Ruiz, M. and Lefranc, M.-P., T cell receptor and
MHC structural data. Nucl. Acids. Res., 32, D208-D210 (2004)].
It must be understood that, without contradictory specification in the present
specification, complementarity-determining regions or CDRs, mean the hypervariable
regions of the heavy and light chains of immunoglobulins as defined according to the
IMGT numbering system.
Nevertheless, CDRs can also be defined according to the Kabat numbering
system (Kabat et al, Sequences of proteins of immunological interest, 5th Ed., U.S.
Department of Health and Human Services, NIH, 1991, and later editions). There are
three heavy chain CDRs and three light chain CDRs. Here, the terms "CDR" and
"CDRs" are used to indicate, depending on the case, one or more, or even all, of the
regions containing the majority of the amino acid residues responsible for the
antibody's binding affinity for the antigen or epitope it recognizes. In order to simplify
the reading of the present application, the CDRs according to Kabat are not defined.
Nevertheless, it would be obvious for the person skilled in the art, using the definition
of the CDRs according to IMGT, to define the CDRs according to Kabat.
In the sense of the present invention, the "identity" or "percentage identity"
between two sequences of nucleic acids or amino acids means the percentage of
identical nucleotides or amino acid residues between the two sequences to be compared,
obtained after optimal alignment, this percentage being purely statistical and the
differences between the two sequences being distributed randomly along their length.
The comparison of two nucleic acid or amino acid sequences is traditionally carried out
by comparing the sequences after having optimally aligned them, said comparison being
able to be conducted by segment or by using an "alignment window". Optimal
alignment of the sequences for comparison can be carried out, in addition to comparison
by hand, by means of the local homology algorithm of Smith and Waterman (1981)
[Ad. App. Math. 2:482], by means of the local homology algorithm of Neddleman and
Wunsch (1970) [J. Mol. Biol. 48:443], by means of the similarity search method of
Pearson and Lipman (1988) [Proc. Natl. Acad. Sci. USA 85:2444] or by means of
computer software using these algorithms (GAP, BESTFIT, FASTA and TFASTA in
the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr.,
Madison, WI, or by the comparison software BLAST NR or BLAST P).
Percentage identity is calculated by determining the number of positions at
which the amino acid nucleotide or residue is identical between the two sequences,
preferably between the two complete sequences, dividing the number of identical
positions by the total number of positions in the alignment window and multiplying the
result by 100 to obtain the percentage identity between the two sequences.
For example, the BLAST program, "BLAST 2 sequences" (Tatusova et al,
"Blast 2 sequences - a new tool for comparing protein and nucleotide sequences",
FEMS Microbiol, 1999, Lett. 174:247-250) available on the site
http://www.ncbi.nlm.nih.gov/gorf/bl2.html, can be used with the default parameters
(notably for the parameters "open gap penalty": 5, and "extension gap penalty": 2; the
selected matrix being for example the "BLOSUM 62" matrix proposed by the program);
the percentage identity between the two sequences to compare is calculated directly by
the program.
For the amino acid sequence exhibiting at least 80%, preferably 85%, 90%, 95%
and 98%> identity with a reference amino acid sequence, preferred examples include
those containing the reference sequence, certain modifications, notably a deletion,
addition or substitution of at least one amino acid, truncation or extension. In the case of
substitution of one or more consecutive or non-consecutive amino acids, substitutions
are preferred in which the substituted amino acids are replaced by "equivalent" amino
acids. Here, the expression "equivalent amino acids" is meant to indicate any amino
acids likely to be substituted for one of the structural amino acids without however
modifying the biological activities of the corresponding antibodies and of those specific
examples defined below.
Equivalent amino acids can be determined either on their structural homology
with the amino acids for which they are substituted or on the results of comparative tests
of biological activity between the various antibodies likely to be generated.
As a non-limiting example, table 1 below summarizes the possible substitutions
likely to be carried out without resulting in a significant modification of the biological
activity of the corresponding modified antibody; inverse substitutions are naturally
possible under the same conditions.
Table 1
A particular aspect of the invention is that the antibody of the ADC, does not
bind to the Insulin receptor (IR). This aspect is of interest as the antibody herein
described will not have any negative impact on the IR, meaning the Insulin metabolism.
In another embodiment, still another advantageous aspect of the antibody of the
ADC of the invention is that it is capable of binding not only to the human IGF-IR but
also to the monkey IGF-IR, and more particularly to the cynomolgus IGF-IR. This
aspect is also of interest as it will facilitate the toxicity assessement required for clinical
trials.
In still another embodiment, the antibody of the ADC of the invention consists
of a monoclonal antibody.
The term "monoclonal antibody" or "Mab" as used herein refers to an antibody
obtained from a population of substantially homogeneous antibodies, i.e. the individual
antibodies of the population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal antibodies are highly
specific, being directed against a single epitope. Such monoclonal antibody may be
produced by a single clone of B cells or hybridoma. Monoclonal antibodies may also
be recombinant, i.e. produced by protein engineering or chemical synthesis.
Monoclonal antibodies may also be isolated from phage antibody libraries. In addition,
in contrast with preparations of polyclonal antibodies which typically include various
antibodies directed against various determinants, or epitopes, each monoclonal
antibody is directed against a single epitope of the antigen.
The monoclonal antibody herein includes murine, chimeric and humanized
antibody, such as described after.
The antibody is preferably derived from an hybridoma of murine origin filed
within the French collection for microorganism cultures (CNCM, Pasteur Institute, 25
rue du Docteur Roux, 75724 Paris Cedex 15, France), said hybridoma being obtained by
the fusion of Balb/C immunized mice splenocytes/lymphocytes and cells of the
myeloma Sp 2/O-Ag 14 cell line.
In an embodiment, the IGF-IR antibody of the ADC of the invention consists of
a murine antibody, then referred as m[name of the antibody].
In an embodiment, the IGF-IR antibody consists of a chimeric antibody, then
referred as c[name of the antibody].
In an embodiment, the IGF-IR antibody consists of a humanized antibody, then
referred as hz[name of the antibody].
For the avoidance of doubt, in the following specification, the expressions "IGFIR
antibody" and "[name of the antibody]" are similar and include (without contrary
specification) the murine, the chimeric and the humanized versions of the said IGF-IR
antibody or of the said "[name of the antibody]". When necessary, the prefix m-
(murine), c- (chimeric) or hz- (humanized) is used.
For more clarity, the following table 2 illustrates the CDR sequences, defined
according to IMGT, for the preferred antibodies.
Table 2
It will be obvious for the man skilled in the art that any combination of 6 CDRs
as above described should be considered as part of the present invention.
As can be observed from this table 2, all the antibodies herein described have the
same sequences for the CDR-H2, CDR-H3 and CDR-L2, this property being of
particular interest as above described.
A specific aspect relates to an ADC wherein the antibody is a murine antibody
characterized in that said antibody also comprises light chain and heavy chain constant
regions derived from an antibody of a species heterologous with the mouse, notably
man.
Another specific aspect relates to an ADC wherein the antibody is a chimeric (c)
antibody characterized in that said antibody also comprises light chain and heavy chain
constant regions derived from an antibody of a species heterologous with the mouse,
notably human.
A chimeric antibody is one containing a natural variable region (light chain and
heavy chain) derived from an antibody of a given species in combination with constant
regions of the light chain and the heavy chain of an antibody of a species heterologous
to said given species.
The chimeric antibodies can be prepared by using the techniques of recombinant
genetics. For example, the chimeric antibody could be produced by cloning recombinant
DNA containing a promoter and a sequence coding for the variable region of a
nonhuman monoclonal antibody, notably murine, and a sequence coding for
heterologous species antibody constant region, preferably human. A chimeric antibody
of the ADC according to the invention coded by one such recombinant gene could be,
for example, a mouse-human chimera, the specificity of this antibody being determined
by the variable region derived from the murine DNA and its isotype determined by the
constant region derived from human DNA.
In a preferred, but not limitative, embodiment, the antibody of the ADC of the
invention is selected from:
a) an antibody comprising a heavy chain variable domain of sequence
SEQ ID No. 13 or any sequence exhibiting at least 80% identity with SEQ ID No. 13
and the three light chain CDRs of sequences SEQ ID Nos. 9, 5 and 11;
b) an antibody comprising a heavy chain variable domain of sequence
SEQ ID No. 14 or any sequence exhibiting at least 80% identity with SEQ ID No. 14
and the three light chain CDRs of sequences SEQ ID Nos. 10, 5 and 11;
c) an antibody comprising a heavy chain variable domain of sequence
SEQ ID No. 15 or any sequence exhibiting at least 80% identity with SEQ ID No. 15
and the three light chain CDRs of sequences SEQ ID Nos. 9, 5 and 12;
d) an antibody comprising a heavy chain variable domain of sequence
SEQ ID No. 16 or any sequence exhibiting at least 80% identity with SEQ ID No. 16
and the three light chain CDRs of sequences SEQ ID Nos. 9, 5 and 11; and
e) an antibody comprising a heavy chain variable domain of sequence
SEQ ID No. 17 or any sequence exhibiting at least 80% identity with SEQ ID No. 17
and the three light chain CDRs of sequences SEQ ID Nos. 9, 5 and 12.
By "any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98%
identity with SEQ ID No. 13 to 17", its is intended to designate the sequences exhibiting
the three heavy chain CDRs SEQ ID Nos. 1, 2 and 3 and, in addition, exhibiting at least
80%, preferably 85%, 90%, 95% and 98%, identity with the full sequence SEQ ID No.
13 to 17 outside the sequences corresponding to the CDRs (i.e. SEQ ID No. 1, 2 and 3).
In another preferred, but not limitative, embodiment, the antibody of the ADC of
the invention is selected from:
a) an antibody comprising a light chain variable domain of sequence
SEQ ID No. 18 or any sequence exhibiting at least 80% identity with SEQ ID No. 18
and the three heavy chain CDRs of sequences SEQ ID Nos. 7, 2 and 3;
b) an antibody comprising a light chain variable domain of sequence
SEQ ID No. 19 or any sequence exhibiting at least 80% identity with SEQ ID No. 19
and the three heavy chain CDRs of sequences SEQ ID Nos. 7, 2 and 3;
c) an antibody comprising a light chain variable domain of sequence
SEQ ID No. 20 or any sequence exhibiting at least 80% identity with SEQ ID No. 20
and the three heavy chain CDRs of sequences SEQ ID Nos. 7, 2 and 3;
d) an antibody comprising a light chain variable domain of sequence
SEQ ID No. 2 1 or any sequence exhibiting at least 80% identity with SEQ ID No. 2 1
and the three heavy chain CDRs of sequences SEQ ID Nos. 8, 2 and 3; and
e) an antibody comprising a light chain variable domain of sequence
SEQ ID No. 22 or any sequence exhibiting at least 80% identity with SEQ ID No. 22
and the three heavy chain CDRs of sequences SEQ ID Nos. 7, 2 and 3.
By "any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98%
identity with SEQ ID No. 18 to 22", its is intended to designate respectively the
sequences exhibiting the three light chain CDRs SEQ ID Nos. 4, 5 and 6 and, in
addition, exhibiting at least 80%, preferably 85%, 90%, 95% and 98% , identity with the
full sequence SEQ ID No. 18 to 22 outside the sequences corresponding to the CDRs
(i.e. SEQ ID No. 4, 5 and 6).
An embodiment of the invention relates to an ADC wherein Ab is an antibody
selected from:
a) an antibody comprising a heavy chain variable domain of sequence
SEQ ID No. 13 or any sequence exhibiting at least 80% identity with SEQ ID No. 13
and a light chain variable domain of sequence SEQ ID No. 18 or any sequence
exhibiting at least 80% identity with SEQ ID No. 18;
b) an antibody comprising a heavy chain variable domain of sequence
SEQ ID No. 14 or any sequence exhibiting at least 80% identity with SEQ ID No. 14
and a light chain variable domain of sequence SEQ ID No. 19 or any sequence
exhibiting at least 80% identity with SEQ ID NO. 19;
c) an antibody comprising a heavy chain variable domain of sequence
SEQ ID No. 15 or any sequence exhibiting at least 80% identity with SEQ ID No. 15
and a light chain variable domain of sequence SEQ ID No. 20 or any sequence
exhibiting at least 80% identity with SEQ ID No. 20;
d) an antibody comprising a heavy chain variable domain of sequence
SEQ ID No. 16 or any sequence exhibiting at least 80% identity with SEQ ID No. 16
and a light chain variable domain of sequence SEQ ID No. 2 1 or any sequence
exhibiting at least 80% identity with SEQ ID No. 21; and
e) an antibody comprising a heavy chain variable domain of sequence
SEQ ID No. 17 or any sequence exhibiting at least 80% identity with SEQ ID No. 17
and a light chain variable domain of sequence SEQ ID No. 22 or any sequence
exhibiting at least 80% identity with SEQ ID No. 22.
Chimeric antibodies herein described can be also characterized by the constant
domain and , more particularly, said chimeric antibodies can be selected or designed
such as, without limitation, IgGl, IgG2, IgG3, IgM, IgA, IgD or IgE. More preferably,
in the context of the present invention, said chimeric antibodies are IgGl or IgG4.
An embodiment of the invention relates to an ADC wherein Ab is a chimeric
antibody comprising variable domains VH and VL as above described in the format
IgGl . More preferably, said chimeric antibody comprises a constant domain for the VH
of sequence SEQ ID No. 43 and a Kappa domain for the VL of sequence SEQ ID No.
45.
An embodiment of the invention relates to an ADC wherein Ab is a chimeric
antibody comprising variable domains VH and VL as above described in the format
IgG4. More preferably, said chimeric antibody comprises a constant domain for the VH
of sequence SEQ ID No. 44 and a Kappa domain for the VL of sequence SEQ ID No.
45.
In another preferred, but not limitative, embodiment, the antibody of the ADC of
the invention is selected from:
a) an antibody comprising or consisting of a heavy chain of sequence
SEQ ID No. 23 or any sequence exhibiting at least 80% identity with SEQ ID No. 23
and a light chain of sequence SEQ ID No. 28 or any sequence exhibiting at least 80%
identity with SEQ ID No. 28;
b) an antibody comprising or consisting of a heavy chain of sequence
SEQ ID No. 24 or any sequence exhibiting at least 80% identity with SEQ ID No. 24
and a light chain of sequence SEQ ID No. 29 or any sequence exhibiting at least 80%
identity with SEQ ID No. 29;
c) an antibody comprising or consisting of a heavy chain of sequence
SEQ ID No. 25 or any sequence exhibiting at least 80% identity with SEQ ID No. 25
and a light chain of sequence SEQ ID No. 30 or any sequence exhibiting at least 80%
identity with SEQ ID No. 30;
d) an antibody comprising or consisting of a heavy chain of sequence
SEQ ID No. 26 or any sequence exhibiting at least 80% identity with SEQ ID No. 26
and a light chain of sequence SEQ ID No. 3lor any sequence exhibiting at least 80%
identity with SEQ ID No. 31; and
e) an antibody comprising or consisting of a heavy chain of sequence
SEQ ID No. 27 or any sequence exhibiting at least 80% identity with SEQ ID No. 27
and a light chain of sequence SEQ ID No. 32 or any sequence exhibiting at least 80%
identity with SEQ ID No. 32.
For more clarity, the following table 3 illustrates the sequences of the VH and
VL, respectively, for the preferred chimeric antibodies.
Table 3
Yet another specific aspect of the present invention relates to an ADC wherein
"Ab" is a humanized antibody characterized in that the constant regions of the light
chain and the heavy chain derived from human antibody are, respectively, the lambda or
kappa region and the gamma- 1, gamma-2 or gamma-4 region.
"Humanized antibodies" means an antibody that contains CDR regions derived
from an antibody of nonhuman origin, the other parts of the antibody molecule being
derived from one (or several) human antibodies. In addition, some of the skeleton
segment residues (called FR) can be modified to preserve binding affinity.
The humanized antibodies or fragments of same can be prepared by techniques
known to a person skilled in the art. Such humanized antibodies are preferred for their
use in methods involving in vitro diagnoses or preventive and/or therapeutic treatment
in vivo. Other humanization techniques, also known to a person skilled in the art, such
as, for example, the "CDR grafting" technique described by PDL in patents EP 0 451
216, EP 0 682 040, EP 0 939 127, EP 0 566 647 or US 5,530,101, US 6,180,370, US
5,585,089 and US 5,693,761. US patents 5,639,641 or 6,054,297, 5,886,152 and
5,877,293 can also be cited.
As a particular embodiment of the invention, and as it will be explicated in more
details in the examples after, it is herein described an antibody consisting of the
hz208F2. Such humanization can also be applied to the other antibodies part of the
present inventioninvention.
In a preferred embodiment, the antibody of the ADC according to the present
invention comprises a heavy chain variable domain (VH) having:
i) the CDR-H1, CDR-H2 and CDR-H3 of sequences SEQ ID Nos. 7, 2 and 3,
respectively, and
ii) the FR1, FR2 and FR3 derived from the human germline IGHV 1-46*01 (SEQ
ID No. 46), and
iii) the FR4 derived from the human germline IGHJ4*01 (SEQ ID No. 48).
In a preferred embodiment, the antibody of the ADC according to the present
invention comprises a light chain variable domain (VL) having:
i) the CDR-L1, CDR-L2 and CDR-L3 of sequences SEQ ID Nos. 9, 5 and 11,
respectively, and
ii) the FRl, FR2 and FR3 derived from the human germline IGKV1-39*01
(SEQ ID No. 47), and
iii) the FR4 derived from the human germline IGKJ4*01 (SEQ ID No. 49).
In a preferred, but not limitative, embodiment of the invention, the antibody
comprises:
a) a heavy chain having CDR-H1, CDR-H2 and CDR-H3 of sequences SEQ ID
Nos. 7, 2 and 3, respectively, and FRl, FR2 and FR3 derived from the human germline
IGHV 1-46*0 1 (SEQ ID No. 46), and the FR4 derived from the human germline
IGHJ4*01 (SEQ ID No. 48); and
b) a light chain having CDR-L1, CDR-L2 and CDR-L3 of sequences SEQ ID
Nos. 9, 5 and 11, respectively, and FRl, FR2 and FR3 derived from the human germline
IGKV1-39*01 (SEQ ID No. 47), and the FR4 derived from the human germlme
IGKJ4*01 (SEQ ID No. 49).
In an embodiment, the antibody of the ADC according to the invention
comprises a heavy chain variable domain (VH) of sequence SEQ ID No. 33 and a light
chain variable domain (VL) of sequence SEQ ID No. 35. Said humanized antibody will
be called thereinafter hz208F2 ("Variant 1" or "Var. 1").
In another embodiment, the antibody of the ADC according to the present
invention comprises a heavy chain variable domain (VH) of sequence SEQ ID No. 33
wherein said sequence SEQ ID No. 33 comprises at least 1 back-mutation selected from
the residues 20, 34, 35, 38, 48, 50, 59, 61, 62, 70, 72, 74, 76, 77, 79, 82 and 95.
By the expressions "back-mutation" or "back mutation" it is meant a mutation or
replacement of the human residue present in the germline by the corresponding residue
initially present in the murine sequence.
In another embodiment, the antibody of the ADC according to the present
invention comprises a heavy chain variable domain (VH) of sequence SEQ ID No. 33
wherein said sequence SEQ ID No. 33 comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16 or 17 back-mutations selected from the residues 20, 34, 35, 38, 48, 50, 59, 61, 62,
70, 72, 74, 76, 77, 79, 82 and 95.
For more clarity, the following table 4 illustrates the preferred back-mutations.
Table 4
In an embodiment, the antibody of the ADC according to the present invention
comprises a light chain variable domain (VL) of sequence SEQ ID No. 35, wherein said
sequence SEQ ID No. 35 comprises at least 1 back-mutation selected from the residues
22, 53, 55, 65, 71, 72, 77 and 87.
In an embodiment, the antibody of the ADC according to the present invention
comprises a light chain variable domain (VL) of sequence SEQ ID No. 35, wherein said
sequence SEQ ID No. 35 comprises 2, 3, 4, 5, 6, 7 or 8 back-mutations selected from
the residues 22, 53, 55, 65, 71, 72, 77 or 87.
In another embodiment, the antibody of the ADC according to the present
invention comprises:
a) a heavy chain variable domain (VH) of sequence SEQ ID No. 33 wherein said
sequence SEQ ID No. 33 comprises at least 1 back-mutation selected from the residues
20, 34, 35, 38, 48, 50, 59, 61, 62, 70, 72, 74, 76, 77, 79, 82 and 95; and
b) a light chain variable domain (VL) of sequence SEQ ID No. 35, wherein said
sequence SEQ ID No. 35 comprises at least 1 back-mutation selected from the residues
22, 53, 55, 65, 71, 72, 77 and 87.
more clarity, the following table 5 illustrates the preferred back-mutations.
Table 5
In such an embodiment, the antibody of the ADC according to the invention
comprises all the back-mutations above mentioned and corresponds to an antibody
comprising a heavy chain variable domain (VH) of sequence SEQ ID No. 34 and a light
chain variable domain (VL) of sequence SEQ ID No. 36. Said humanized antibody will
be called thereinafter hz208F2 ("Variant 3" or "Var. 3").
In another embodiment, all the humanized forms comprised between the Variant
1 and the Variant 3 are also encompassed by the present invention. In other words, the
antibody according to the invention corresponds to an antibody comprising a heavy
chain variable domain (VH) of "consensus" sequence SEQ ID No. 4 1 and a light chain
variable domain (VL) of "consensus" sequence SEQ ID No. 42. Said humanized
antibody, as a whole, will be called thereinafter hz208F2 ("Variant2" or "Var.2").
In a preferred, but not limitative, embodiment, the antibody of the ADC of the
invention is selected from:
a) an antibody comprising a heavy chain variable domain of sequence
SEQ ID No. 33 or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and
98% identity with SEQ ID No. 33 and the three light chain CDRs of sequences SEQ ID
Nos. 9, 5 and 11; and
b) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 34
or any sequence exhibiting at least 80%>, preferably 85%, 90%>, 95% and 98%> identity
with SEQ ID No. 34 and the three light chain CDRs of sequences SEQ ID Nos. 9, 5 and
11.
By "any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98%
identity with SEQ ID No. 33 or 34", its is intended to designate the sequences
exhibiting the three heavy chain CDRs SEQ ID Nos. 1, 2 and 3 and, in addition,
exhibiting at least 80%, preferably 85%, 90%, 95% and 98%, identity with the full
sequence SEQ ID No. 33 or 34 outside the sequences corresponding to the CDRs (i.e.
SEQ ID Nos. 1, 2 and 3).
If not indicated in the concerned paragraphs, in the present description, by any
sequence or by a sequence exhibiting at least 80%> with a particular sequence, it must be
understood that said sequence exhibits at least 80%> and preferably 85%, 90%, 95% and
9 8% identity with the referenced sequence.Whether these sequences contain CDR
sequences, its is intended to designate that the sequences exhibiting at least these CDRs
identically to the reference sequence CDRs, the 80%, preferably 85%, 90%, 95% and
98%, identity with the full sequence having to be calculated for the remaining sequence
located outside the sequences corresponding to these CDRs.
In a preferred, but not limitative, embodiment, the antibody of the invention is
selected from:
a) an antibody comprising a light chain variable domain of sequence
SEQ ID No. 35 or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and
9 8% identity with SEQ ID No. 35 and the three heavy chain CDRs of sequences SEQ
ID Nos. 7, 2 and 3; and
b) an antibody comprising a light chain variable domain of sequence
SEQ ID No. 36 or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and
98% identity with SEQ ID No. 36 and the three heavy chain CDRs of sequences SEQ
ID Nos. 7, 2 and 3.
By "any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98%
identity with SEQ ID No. 35 or 36", its is intended to designate the sequences
exhibiting the three light chain CDRs SEQ ID Nos. 4, 5 and 6 and, in addition,
exhibiting at least 80%, preferably 85%, 90%, 95% and 98%, identity with the full
sequence SEQ ID No. 35 or 36 outside the sequences corresponding to the CDRs (i.e.
SEQ ID Nos. 4, 5 and 6).
Humanized antibodies herein described can be also characterized by the constant
domain and, more particularly, said humanized antibodies can be selected or designed
such as, without limitation, IgGl, IgG2, IgG3, IgM, IgA, IgD or IgE. More preferably,
in the context of the present invention, said humanized antibodies are IgGl or IgG4.
An embodiment of the invention relates to an ADC wherein "Ab" is a
humanized antibody comprising variable domains VH and VL as above described in the
format IgGl. More preferably, said humanized antibody comprises a constant domain
for the VH of sequence SEQ ID No. 43 and a Kappa domain for the VL of sequence
SEQ ID No. 45.
An embodiment of the invention relates to an ADC wherein "Ab" is a
humanized antibody comprising variable domains VH and VL as above described in the
format IgG4. More preferably, said humanized antibody comprises a constant domain
for the VH of sequence SEQ ID No. 44 and a Kappa domain for the VL of sequence
SEQ ID No. 45.
Still another embodiment of the invention relates to an ADC wherein "Ab" is an
antibody selected from:
a) an antibody comprising or consisting of a heavy chain of sequence
SEQ ID No. 37 or any sequence exhibiting at least 80% identity with SEQ ID No. 37
and a light chain of sequence SEQ ID No. 39 or any sequence exhibiting at least 80%
identity with SEQ ID No. 39; and
b) an antibody comprising or consisting of a heavy chain of sequence
SEQ ID No. 38 or any sequence exhibiting at least 80% identity with SEQ ID No. 38
and a light chain of sequence SEQ ID No. 40 or any sequence exhibiting at least 80%
identity with SEQ ID No. 40.
For more clarity, the following table 6a illustrates non limitative examples of
sequences of the VH and VL for the variant 1 (Var. 1) and the variant 3 (Var. 3) of the
humanized antibody hz208F2. It also comprises the consensus sequence for the variant
2 (Var. 2).
Table 6a
In another preferred, but not limitative, embodiment, the antibody of the ADC of
the invention is selected from:
a) an antibody comprising a heavy chain variable domain of sequence selected
from SEQ ID Nos. 56, 62, 64, 66, 68, 70, 72, 74, 76, 78 and 80 or any sequence with at
least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID No.56, 62, 64,
66, 68, 70, 72, 74, 76, 78 and 80; and the three light chain CDRs of sequences SEQ ID
Nos. 9, 5 and 1;
b) an antibody comprising a light chain variable domain of sequence selected
from SEQ ID Nos. 57 or 60 or any sequence with at least 80%, preferably 85%, 90%,
95% and 98% identity with SEQ ID Nos. 57 or 60; and the three heavy chain CDRs of
sequences SEQ ID Nos. 7, 2 and 3; and
c) an antibody comprising a heavy chain variable domain of sequence selected
from SEQ ID Nos. 56, 62, 64, 66, 68, 70, 72, 74, 76, 78 and 80 or any sequence with at
least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID Nos.56, 62, 64,
66, 68, 70, 72, 74, 76, 78 and 80; and a light chain variable domain of sequence selected
from SEQ ID Nos. 57 or 60 or any sequence with at least 80%, preferably 85%, 90%,
95% and 98% identity with SEQ ID Nos. 57 or 60.
Still another embodiment of the invention relates to an ADC wherein "Ab"
antibody selected from:
a) an antibody comprising a heavy chain of sequence SEQ ID Nos. 56, 62, 64,
66, 68, 70, 72, 74, 76, 78 and 80 or any sequence exhibiting at least 80%> identity with
SEQ ID No. 56, 62, 64, 66, 68, 70, 72, 74, 76, 78 or 80, and a light chain of sequence
SEQ ID No. 57 or any sequence exhibiting at least 80%> identity with SEQ ID No. 57;
and
b) an antibody comprising a heavy chain of sequence SEQ ID Nos. 56, 64, 68
and 78 or any sequence exhibiting at least 80%> identity with SEQ ID No. 56, 64, 68 or
78 and a light chain of sequence SEQ ID No. 60, or any sequence exhibiting at least
80% identity with SEQ ID No. 60.
Still another embodiment of the invention relates to an ADC wherein Ab is an
antibody selected from:
a) an antibody comprising or consisting of a heavy chain of sequence
SEQ ID No. 58 or any sequence exhibiting at least 80% identity with SEQ ID No. 58
and a light chain of sequence SEQ ID No. 59 or any sequence exhibiting at least 80%
identity with SEQ ID No. 59;
b) an antibody comprising or consisting of a heavy chain of sequence
SEQ ID No. 58 or any sequence exhibiting at least 80% identity with SEQ ID No. 58
and a light chain of sequence SEQ ID No. 6 1 or any sequence exhibiting at least 80%
identity with SEQ ID No. 61;
c) an antibody comprising or consisting of a heavy chain of sequence
SEQ ID No. 63 or any sequence exhibiting at least 80% identity with SEQ ID No. 63
and a light chain of sequence SEQ ID No. 59 or any sequence exhibiting at least 80%
identity with SEQ ID No. 59;
d) an antibody comprising or consisting ofa heavy chain of sequence
SEQ ID No. 65 or any sequence exhibiting at least 80% identity with SEQ ID No. 65
and a light chain of sequence SEQ ID No. 59 or any sequence exhibiting at least 80%
identity with SEQ ID No. 59;
e) an antibody comprising or consisting of a heavy chain of sequence
SEQ ID No. 65 or any sequence exhibiting at least 80% identity with SEQ ID No. 65
and a light chain of sequence SEQ ID No. 6 1 or any sequence exhibiting at least 80%
identity with SEQ ID No. 61;
f an antibody comprising or consisting of a heavy chain of sequence
SEQ ID No. 67 or any sequence exhibiting at least 80% identity with SEQ ID No. 67
and a light chain of sequence SEQ ID No. 59 or any sequence exhibiting at least 80%
identity with SEQ ID No. 59;
g) an antibody comprising or consisting of a heavy chain of sequence
SEQ ID No. 69 or any sequence exhibiting at least 80% identity with SEQ ID No. 69
and a light chain of sequence SEQ ID No. 59 or any sequence exhibiting at least 80%
identity with SEQ ID No. 59;
h) an antibody comprising or consisting of a heavy chain of sequence
SEQ ID No. 69 or any sequence exhibiting at least 80% identity with SEQ ID No. 69
and a light chain of sequence SEQ ID No. 6 1 or any sequence exhibiting at least 80%
identity with SEQ ID No. 61;
i) an antibody comprising or consisting of a heavy chain of sequence
SEQ ID No. 7 1 or any sequence exhibiting at least 80% identity with SEQ ID No. 7 1
and a light chain of sequence SEQ ID No. 59 or any sequence exhibiting at least 80%
identity with SEQ ID No. 59;
j) an antibody comprising or consisting of a heavy chain of sequence
SEQ ID No. 73 or any sequence exhibiting at least 80% identity with SEQ ID No. 73
and a light chain of sequence SEQ ID No. 59 or any sequence exhibiting at least 80%
identity with SEQ ID No. 59;
k) an antibody comprising or consisting of a heavy chain of sequence
SEQ ID No. 75 or any sequence exhibiting at least 80% identity with SEQ ID No. 75
and a light chain of sequence SEQ ID No. 59 or any sequence exhibiting at least 80%
identity with SEQ ID No. 59;
1) an antibody comprising or consisting of a heavy chain of sequence
SEQ ID No. 77 or any sequence exhibiting at least 80% identity with SEQ ID No. 77
and a light chain of sequence SEQ ID No. 59 or any sequence exhibiting at least 80%
identity with SEQ ID No. 59;
m) an antibody comprising or consisting of a heavy chain of sequence
SEQ ID No. 79 or any sequence exhibiting at least 80% identity with SEQ ID No. 79
and a light chain of sequence SEQ ID No. 59 or any sequence exhibiting at least 80%
identity with SEQ ID No. 59;
n) an antibody comprising or consisting of a heavy chain of sequence
SEQ ID No. 79 or any sequence exhibiting at least 80% identity with SEQ ID No. 79
and a light chain of sequence SEQ ID No. 6 1 or any sequence exhibiting at least 80%
identity with SEQ ID No. 61; and
o) an antibody comprising or consisting of a heavy chain of sequence
SEQ ID No. 8 1 or any sequence exhibiting at least 80% identity with SEQ ID No. 8 1
and a light chain of sequence SEQ ID No. 59 or any sequence exhibiting at least 80%
identity with SEQ ID No. 59.
In other words, the invention relates to an ADC wherein Ab is an antibody
comprising:
a) a heavy chain of sequence selected from SEQ ID Nos. 58, 63, 65, 67, 69, 71, 73, 75,
77, 79 and 8 1 or any sequence with at least 80%> identity with SEQ ID Nos. 58, 63, 65,
67, 69, 71, 73, 75, 77, 79 and 81; and
b) a light chain of sequence selected from SEQ ID Nos. 59 and 6 1 or any sequence with
at least 80% identity with SEQ ID Nos. 59 and 6 1.
For more clarity, the following table 6b illustrates non limitative examples of
sequences of the VH and VL (vaiable domain and full lengh) for different variants of
the humanized antibody hz208F2.
Table 6b
Heavy Chain Light chain SEQ ID NO.
Variable domain (VH) 56
hz208F2 Variable domain (VL) 57
H037/L01 8 Full length 58
Full length 59
Variable domain (VH) 56
Hz208F2 Variable domain (VL) 60
H037/L021 Full length 58
Full length 6 1
Variable domain (VH) 62
Hz208F2 Variable domain (VL) 57
H047/L01 8 Full length 63
Full length 59
Variable domain (VH) 64
Hz208F2 Variable domain (VL) 57
H049/L01 8 Full length 65
Full length 59
Variable domain (VH) 64
Hz208F2 Variable domain (VL) 60
H049/L021 Full length 65
Full length 6 1
Variable domain (VH) 66
Hz208F2 Variable domain (VL) 57
H05 1/L01 8 Full length 67
Full length 59
Variable domain (VH) 68
Hz208F2 Variable domain (VL) 57
H052/L01 8 Full length 69
Full length 59
Variable domain (VH) 68
Hz208F2 Variable domain (VL) 60
H052/L021 Full length 69
Full length 6 1
Variable domain (VH) 70
Hz208F2 Variable domain (VL) 57
H057/L01 8 Full length 7 1
Full length 59
Variable domain (VH) 72
Hz208F2 Variable domain (VL) 57
H068/L018 Full length 73
Full length 59
Variable domain (VH) 74
Hz208F2 Variable domain (VL) 57
H070/L018 Full length 75
Full length 59
Variable domain (VH) 76
Hz208F2 Variable domain (VL) 57
H071/L018 Full length 77
Full length 59
Variable domain (VH) 78
Hz208F2 Variable domain (VL) 57
H076/L018 Full length 79
Full length 59
Variable domain (VH) 78
Hz208F2 Variable domain (VL) 60
H076/L021 Full length 79
Full length 6 1
Variable domain (VH) 80
Hz208F2 Variable domain (VL) 57
H077/L018 Full length 8 1
Full length 59
Another aspect of the present invention is an ADC wherein Ab is an antibody
selected from i) an antibody produced by the hybridoma 1-4757, 1-4773, 1-4775, 1-4736
or 1-4774 deposited at the CNCM, Institut Pasteur France on the 30 May 2013, 26 June
2013, 26 June 2013, 24 April 2013 and 26 June 2013, respectively, or ii) an antibody
which competes for binding to IGF-1R with the antibody of i); or iii) an antibody which
binds to the same epitope of IGF-1R as does the antibody of i).
Indeed, it is described herein the murine hybridoma selected from the hybridoma
1-4757, 1-4773, 1-4775, 1-4736 and 1-4774 deposited at the CNCM, Institut Pasteur
France on the 30 May 2013, 26 June 2013, 26 June 2013, 24 April 2013 and 26 June
2013, respectively.
It is also described the isolated nucleic acid coding for an antibody, or for an
antigen binding fragment thereof, according to the invention.
The terms "nucleic acid", "nucleic sequence", "nucleic acid sequence",
"polynucleotide", "oligonucleotide", "polynucleotide sequence" and "nucleotide
sequence", used interchangeably in the present description, mean a precise sequence of
nucleotides, modified or not, defining a fragment or a region of a nucleic acid,
containing unnatural nucleotides or not, and being either a double-strand DNA, a singlestrand
DNA or transcription products of said DNAs.
These sequences have been isolated and/or purified, i.e., they were sampled
directly or indirectly, for example by a copy, their environment having been at least
partially modified. Isolated nucleic acids obtained by recombinant genetics, by means,
for example, of host cells, or obtained by chemical synthesis should also be mentioned
here.
It is alsoalso described vector comprising a nucleic acid coding for an antibody,
or for an antigen binding fragment thereof, of the ADC according to the invention,
particularly cloning and/or expression vectors that contain such a nucleotide sequence.
The vectors preferably contain elements which allow the expression and/or the
secretion of nucleotide sequences in a given host cell. The vector thus may contain a
promoter, translation initiation and termination signals, as well as suitable transcription
regulation regions. It must be able to be maintained in a stable manner in the host cell
and may optionally have specific signals which specify secretion of the translated
protein. These various elements are selected and optimized by a person skilled in the art
according to the host cell used. For this purpose, the nucleotide sequences can be
inserted in self-replicating vectors within the chosen host or be integrative vectors of the
chosen host.
The vectors are, for example, vectors of plasmid or viral origin. They are used to
transform host cells in order to clone or express the nucleotide sequences of the
invention.
Such vectors are prepared by methods typically used by a person skilled in the
art and the resulting clones can be introduced into a suitable host by standard methods
such as lipofection, electroporation, conjugation, heat shock or chemical methods.
These isolated host cells are transformed by or comprising a vector as above
described.
The host cell can be selected among prokaryotic or eukaryotic systems such as
bacterial cells, for example, but also yeast cells or animal cells, notably mammal cells
(with the exception of human). Insect or plant cells can also be used.
It is also disclosed method for the production of an antibody of the ADC
according to the invention, or an antigen binding fragment thereof, wherein said method
comprises the following steps:
a) the culture in a medium with the suitable culture conditions for a host cell as
above disclosed; and
b) the recovery of the antibody thus produced from the culture medium or from
said cultured cells.
The transformed cells are of use in methods for the preparation of recombinant
antibodies of the ADC according to the invention. Methods for the preparation of
antibodies in recombinant form using a vector and/or a cell transformed by a vector as
above disclosed, are also comprised in the present specification. Preferably, a cell
transformed by a vector as above described is cultured under conditions that allow the
expression of the aforesaid antibody and recovery of said antibody.
As already mentioned, the host cell can be selected among prokaryotic or
eukaryotic systems. In particular, it is possible to identify the nucleotide sequences that
facilitate secretion in such a prokaryotic or eukaryotic system. A vector as above
disclosed carrying such a sequence can thus be used advantageously for the production
of recombinant proteins to be secreted. Indeed, the purification of these recombinant
proteins of interest will be facilitated by the fact that they are present in the supernatant
of the cellular culture rather than inside host cells.
The antibody of the ADC of the present invention can also be prepared by
chemical synthesis. One such method of preparation is also an object of the invention. A
person skilled in the art knows methods for chemical synthesis, such as solid-phase
techniques or partial solid-phase techniques, by condensation of fragments or by
conventional synthesis in solution. Polypeptides obtained by chemical synthesis and
capable of containing corresponding unnatural amino acids can be also cited.
The antibody likely to be obtained by the method above described are also
comprised in the present invention.
According to a particular aspect, the invention concerns an ADC wherein AB is
an antibody, or an antigen binding fragment thereof, as above described for use as an
addressing vehicle for delivering a cytotoxic agent at a host target site, said host target
site consisting of an epitope localized into IGF-IR, preferably the IGF-IR extracellular
domain, more preferably the human IGF-IR (SEQ ID No. 50) and still more preferably
the human IGF-IR extracellular domain (SEQ ID No. 51), and still more preferably to
the N-terminal of the human IGF-IR extracellular domain (SEQ ID No. 52), or any
natural variant sequence thereof.
In a preferred embodiment, said host target site is a target site of a mammalian
cell, more preferably of a human cell, more preferably cells which naturally or by way
of genetic recombination, express IGF-IR.
In a more embodiment, said host target site is a target site of a cell of patient,
preferably human, having a cancer, preferably an IGF-IR expressing cancer, or IGF-IR
related cancers.
IGF-IR expressing cancers or IGF-IR related cancers include particularly
cancers wherein the tumoral cells express or over-express whole or part of the IGF-IR
at their surface.
II - The drug (D)
The drug moiety according to the invention has the following formula (II)
where:
- R2 is COOH, COOCH3 or thiazolyl (such as thiazol-2-yl),
- R 3 is H or a (Ci-C )alkyl (such as methyl), in particular a (Ci-C )alkyl group,
- R 9 is H or (Ci-C6)alkyl (such as methyl),
- m is an integer comprised between 1 and 8, and
- the wavy line indicates the point of attachment to L.
By "alkyl" in the present invention is meant a straight-chain or branched,
saturated hydrocarbon chain. For example, mention can be made of methyl, ethyl,
propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl or hexyl groups.
By "(Cx- Cy)alkyl" in the present invention is meant an alkyl chain such as
defined above comprising x to y carbon atoms. Therefore, a (Ci-C )alkyl group is an
alkyl chain having 1 to 6 carbon atoms.
The (Ci-C )alkyl is advantageously a (Ci-C4)alkyl, peferably a (Ci-C 2)alkyl.
Among the compounds of the invention, one particularly appreciated class of
drug moieties corresponds to the formula (II) drug moieties in which R2 represents a
COOH group.
Another particularly appreciated class of moieties corresponds to the formula
(II) moieties in which R2 is a thiazole (in particular a thiazol-2-yl group).
Another class of particularly appreciated moieties corresponds to the formula
(II) moieties in which R2 is COOMe.
According to one particular embodiment of the present invention, R2 is more
particularly a COOH, COOMe or thiazol-2-yl group.
According to a first preferred embodiment, R2 is COOH.
According to a second preferred embodiment, R2 is COOMe.
R3 particularly represents a (Ci-C6)alkyl, advantageously a methyl group.
m is an integer comprised between 1 and 8, in particular between 1 and 6,
advantageously between 1 and 4, preferably is 1 or 2.
In a preferred embodiment, R2 is COOH, R 3 is a methyl group and m is 1 or 2.
Among the drug moieties of the invention, one particularly appreciated class of
drug moieties corresponds to the formula (II) drug moieties in which R is a methyl
group or a hydrogen.
In a preferred embodiment:
- R2 is COOH, R3 is a methyl group, R9 is a methyl group and m is 1 or 2, or
- R2 is COOH, R3 is a methyl group, R9 is a hydrogen and m is 1 or 2.
According to a preferred embodiment, the NR9 group is located on the phenyl ring
in a para position in relation to the (CH2)m group.
Advantageously, the drug moiety is chosen from among the following moieties:

Preparation of the drug (offormula DH):
The drug can be prepared using the general methods described in the following
synthesis schemes, optionally supplemented by any standard operation when needed
that is described in the literature or well known to persons skilled in the art, or described
in the examples in the experimental part hereof.
Scheme 1
Scheme 1 illustrates the first general method which can be used to prepare the
drug. In the above such as previously defined
for formula II, R4 R 4a represents a R4 group
such as previously defined optionally in protected form and G is a protective group.
The first step consists of the condensing of compound (II), protected on its
amine function by a protective group G, with compound (III). X may represent a leaving
group such as a chlorine. In this case the first step consists of the reaction between an
acid chloride and an amine. This reaction can be conducted using methods and
techniques well known to those skilled in the art. In one particularly appreciated
method, the two entities are caused to react in the presence of an organic or inorganic
base e.g. Et3N, iPr2NEt, pyridine, NaH, Cs2C0 3, K2C0 3in a solvent such as THF,
dichloromethane, DMF, DMSO, at a temperature notably between -20°C and 100°C. X
may also be a hydroxyl (OH). In this case, the first step is a condensation reaction
between the carboxylic acid (II) and the amine (III). This reaction can be performed
following methods and techniques well known to skilled persons. In one particularly
appreciated method, these two entities are caused to react in the presence of a coupling
agent such as l-(3-dimethylaminopropyl)-3-ethyl-carbodiimide (EDC), 3-hydroxyl,
2,3-benzotriazin-4(3H)-one, a tertiary amine such as diisopropylethylamine, in a
polar aprotic solvent such as dichloromethane or DMF, at a temperature notably
between -15°C and 40°C. In another particularly appreciated method, these two entities
are caused to react in the presence of diethyl phosphorocyanidate (DEPC), a tertiary
amine such as triethylamine, in a polar aprotic solvent such as dichloromethane or
DMF, at a temperature of between -15°C and 40°C. Another particularly appreciated
method consists of causing these two entities to react in the presence of 0-(7-
azabenzotriazol-l-yl)-l ,1,3,3-tetramethyl-uroniumhexafluorophosphate (HATU),a
tertiary amine such as diisopropylethylamine, in a polar aprotic solvent such as
dichloromethane or DMF, at a temperature of between-15°C and 100°C.
After deprotection of the intermediate using techniques well known to those
skilled in the art (« Protective Groups in Organic Synthesis », T.W. Greene, John Wiley
& Sons, 2006 and « Protecting Groups », P.J. Kocienski, Thieme Verlag, 1994),
compound (IV) can be condensed with compound (V) following the methods and
techniques described above to lead to compound (VI) after a deprotection step. This
compound can then, after condensation with the intermediate (VII) and optional
deprotection, lead to the formation of the drug. Compound (VI) can also be coupled
with a compound (VIF) in whichR' 3 is a precursor of R , in particular an R3 group
protected by a protective group. Coupling followed by deprotection of group R' to lead
to R can be carried out following the same procedures as described previously.
Scheme 2
Scheme 2 illustrates the second general method which can be used to prepare the
. In the above general formulas, G is a pro and R 4a are
such as previously defined, and R4b represents
At the first step, compound (IX) protected on its amine function by a protective
group G is condensed with compound (VI). X may represent a leaving group e.g. a
chlorine. In this case, the first step consists of the reaction between an acid chloride and
an amine. This reaction can be performed using methods and techniques well known to
persons skilled in the art. In one particularly appreciated method the two entities are
caused to react in the presence of an organic or inorganic base such as Et3N, iPr2NEt,
pyridine, NaH, Cs2C0 3, K2C0 3 in a solvent such as THF, dichloromethane, DMF,
DMSO at a temperature notably between -20° and 100°C. X may also represent a
hydroxyl. In this case, the first step is a condensation reaction between the carboxylic
acid (IX) and the amine (VI). This reaction can be conducted following methods and
techniques well known to skilled persons. In one particularly appreciated method, the
two entities are caused to react in the presence of l-(3-dimethylaminopropyl)-3-ethylcarbodiimide
(EDC), 3-hydroxy-l,2,3-benzotriazin-4(3H)-one, a tertiary amine such
as diisopropylethylamine, in a polar aprotic solvent such as dichloromethane or DMF, at
a temperature notably between -15°C and 40°C. In another particularly appreciated
method, these two entities are caused to react in the presence of diethyl
phosphorocyanidate (DEPC), a tertiary amine such as triethylamine, in a polar aprotic
solvent such as dichloromethane or DMF, at a temperature notably between -15°C and
40°C.
After deprotection of the intermediate, using techniques well known to skilled
persons, the obtained compound (VIII) can lead to the drug after reaction with R4Y. In
this case, Y is a leaving group such as CI, Br, I, OS0 2CH3, OS0 2CF3 or O-Tosyl. The
reaction is conducted in the presence of an organic or inorganic base such as Et3N,
iPr2NEt, NaH, Cs2C0 3, K2C0 3, in a polar anhydrous solvent such as dichloromethane,
THF, DMF, DMSO at a temperature notably between -20° and 100°C. In another
particularly appreciated method, compound (VIII) is caused to react with an aldehyde of
formula R4b-CHO where R4b corresponds to a precursor of R4. In this case, the reaction
is a reductive amination in the presence of a reducing agent such as NaBH 4, NaBH 3CN,
NaBH(OAc) 3, in a polar solvent such as 1,2-dichloroethane, dichloromethane, THF,
DMF, MeOH, in the optional presence of titanium isopropoxide (IV), at a pH which can
be controlled by the addition of an acid such as acetic acid at a temperature notably
between-20°C and 100°C.
In the foregoing synthesis schemes, a drug may lead to another drug after an
additional reaction step such as saponification for example using methods well known
to skilled persons whereby an R2 group representing an ester (COOMe), is changed to
an R2 group representing a carboxylic acid (COOH).
If it is desired to isolate a drug containing at least one base function in the state
of an acid addition salt, this is possible by treating the free base of the drug (containing
at least one base function) with a suitable acid, preferably in equivalent quantity. The
suitable acid may in particular be trifluoroacetic acid.
III - The linker (L)
"Linker", "Linker Unit", "L" or "link" means, in the present invention, a
chemical moiety comprising a covalent bond or a chain of atoms that covalently
attaches an antibody to at least one drug.
Linkers may be made using a variety of bifunctional protein coupling agents
such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(Nmaleimidomethyl)
cyclohexane-l-carboxylate (SMCC), iminothiolane (IT), bifunctional
derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as
disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-
(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-
diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-
dinitrobenzene). Carbon- 14-labeled l-isothiocyanatobenzyl-3-methyldiethylene
triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation
of cyctotoxic agents to the addressing system. Other cross-linker reagents may be
BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC,
SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB,
sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate)
which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, 111.,
U.S. A).
The linker may be a "non cleavable" or "cleavable".
In a preferred embodiment, it consists in a "cleavable linker" facilitating release
of the drug in the cell. For example, an acid-labile linker, peptidase-sensitive linker,
photolabile linker, dimethyl linker or disulfide- containing linker may be used. The
linker is, in a preferred embodiment, cleavable under intracellular conditions, such that
cleavage of the linker releases the drug from the antibody in the intracellular
environment.
For example, in some embodiments, the linker is cleavable by a cleaving agent
that is present in the intracellular environment (e.g., within a lysosome or endosome or
caveolea). The linker can be, for example, a peptidyl linker that is cleaved by an
intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or
endosomal protease. Typically, the peptidyl linker comprises at least two successive
amino acids or at least three successive amino acids or is at least two amino acids long
or at least three amino acids long. Cleaving agents can include cathepsins B and D and
plasmin, all o f which are known to hydrolyze dipeptide drug derivatives resulting in the
release of active drug inside target cells. For example, a peptidyl linker that is cleavable
by the thiol-dependent protease cathepsin-B, which is highly expressed in cancerous
tissue, can be used (e.g., a linker comprising or being Phe-Leu or Gly-Phe-Leu-Gly). In
specific embodiments, the peptidyl linker cleavable by an intracellular protease
comprises or is Val-Cit or Phe-Lys. One advantage of using intracellular proteolytic
release of the drug is that the drug is typically attenuated when conjugated and the
serum stabilities of the conjugates are typically high.
In other embodiments, the cleavable linker is pH-sensitive, i.e., sensitive to
hydrolysis at certain pH values. Typically, the pH-sensitive linker is hydrolyzable under
acidic conditions. For example, an acid-labile linker that is hydrolyzable in the
lysosome (e.g., a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide,
orthoester, acetal, ketal, or the like) can be used. Such linkers are relatively stable under
neutral pH conditions, such as those in the blood, but are unstable at below pH 5.5 or
5.0, the approximate pH of the lysosome. In certain embodiments, the hydrolyzable
linker is a thioether linker (such as, e.g., a thioether attached to the drug via an
acylhydrazone bond).
In yet other embodiments, the linker is cleavable under reducing conditions (e.g.,
a disulfide linker). A variety of disulfide linkers are known in the art, including, for
example, those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate),
SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-
pyridyldithio)butyrate) and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-
(2-pyridyl-dithio)toluene).
In certain preferred embodiments, the linker unit may have the following general
formula:
-(T)a-(W)w-(Y) -
wherein:
T is a stretcher unit;
a is 0 or 1;
W is an amino acid unit;
w is an integer ranging from 0 to 12;
Y is a spacer unit;
y is 0, 1 or 2.
The stretcher unit (T), when present, links the antibody to an amino acid unit
(W) when present, or to the spacer unit when present, or directly to the drug. Useful
functional groups that can be present on the antibody, either naturally or via chemical
manipulation, include sulfhydryl, amino, hydroxyl, the anomeric hydroxyl group of a
carbohydrate, and carboxyl. Suitable functional groups are sulfhydryl and amino.
Sulfhydryl groups can be generated by reduction of the intramolecular disulfide bonds
of the antibody, if present. Alternatively, sulfhydryl groups can be generated by reaction
of an amino group of a lysine moiety of the antibody with 2-iminothiolane or other
sulfhydryl generating reagents. In specific embodiments, the antibody is engineered to
carry one or more lysines. More preferably, the antibody can be engineered to carry one
or more Cysteines (cf. ThioMabs).
In certain specific embodiments, the stretcher unit forms a bond with a sulfur
atom of the antibody. The sulfur atom can be derived from a sulfhydryl (-SH) group of a
reduced antibody.
In certain other specific embodiments, the stretcher unit is linked to the antibody
via a disulfide bond between a sulfur atom of the antibody and a sulfur atom of the
stretcher unit.
In other specific embodiments, the reactive group of the stretcher contains a
reactive site that can be reactive to an amino group of the antibody. The amino group
can be that of an arginine or a lysine. Suitable amine reactive sites include, but are not
limited to, activated esters (such as succinimide esters, 4-nitrophenyl esters,
pentafluorophenyl esters), anhydrides, acid chlorides, sulfonyl chlorides, isocyanates
and isothiocyanates.
In yet another aspect, the reactive function of the stretcher contains a reactive
site that is reactive to a modified carbohydrate group that can be present on the
antibody. In a specific embodiment, the antibody is glycosylated enzymatically to
provide a carbohydrate moiety or is naturally glycosylated. The carbohydrate may be
mildly oxidized with a reagent such as sodium periodate and the resulting carbonyl unit
of the oxidized carbohydrate can be condensed with a stretcher that contains a
functionality such as a hydrazide, an oxime, a reactive amine, a hydrazine, a
thiosemicarbazide, a hydrazine carboxylate, or an arylhydrazide.
According to a particular embodiment, the stretcher unit has the following
formula:
wherein
L2 is (C4 -Cio)cycloalkyl-carbonyl, (C2-C )alkyl or (C2-C )alkyl-carbonyl (the cycloalkyl
or alkyl moieties being linked to the nitrogen atom of the maleimide moiety),
the asterisk indicates the point of attachment to the amino acid unit, if present, to the
spacer unit, if present, or to the drug D, and
the wavy line indicates the point of attachment to the antibody Ab.
By "(C4 -Cio)cycloalkyl" in the present invention is meant a hydrocarbon cycle
having 4 to 10 carbon atoms including, but not limited to, cyclopentyl, cyclohexyl and
the like.
L2 can be advantageously (C2 -C6 )alkyl-carbonyl such as a pentyl-carbonyl of the
following formula:
wherein
the asterisk indicates the point of attachment to the amino acid unit, if present, to the
spacer unit, if present, or to the drug D; and
the wavy line indicates the point of attachment to the nitrogen atom of the maleimide
moiety.
The amino acid unit (W), when present, links the stretcher unit (T) if present, or
otherwise the antibody to the spacer unit (Y) if the spacer unit is present, or to the drug
if the spacer unit is absent.
As above mentioned, (W)w is absent (w = 0) or may be a dipeptide, tripeptide,
tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide,
decapeptide, undecapeptide or dodecapeptide unit, wherein the amino acids forming the
peptides can be different from one another.
Thus (W)w can be represented by the following formula:
(Wl) wi(W2)w2(W3)W3(W4)w4(W5)W5, wherein each Wl to W5 represents, independently
from one another, an amino acid unit and each wl to w5 is 0 or 1.
In some embodiments, the amino acid unit (W)w may comprise amino acid
residues such as those occurring naturally, as well as minor amino acids and nonnaturally
occurring amino acid analogs, such as citrulline.
The amino acid residues of the amino acid unit (W)w include, without limitation,
alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline,
lysine protected or not with acetyl or formyl, arginine, arginine protected or not with
tosyl or nitro groups, histidine, ornithine, ornithine protected with acetyl or formyl, and
citrulline. Exemplary amino acid linker components include preferably a dipeptide, a
tripeptide, a tetrapeptide or a pentapeptide, notably a dipeptide or a tripeptide.
Exemplary dipeptides include: Val-Cit, Ala-Val, Ala-Ala, Val-Ala, Lys-Lys,
Cit-Cit, Val-Lys, Ala-Phe, Phe-Lys, Ala-Lys, Phe-Cit, Leu-Cit, lle-Cit, Trp-Cit, Phe-
Ala, Phe-N -tosyl-Arg, Phe-N -Nitro-Arg.
Exemplary tripeptides include: Val-Ala-Val, Ala-Asn-Val, Val-Leu-Lys, Ala-
Ala-Asn, Phe-Phe-Lys, Gly-Gly-Gly, D-Phe-Phe-Lys, Gly-Phe-Lys.
Exemplary tetrapeptide include: Gly-Phe-Leu-Gly (SEQ ID NO. 53), Ala-Leu-
Ala-Leu (SEQ ID NO. 54).
Exemplary pentapeptide include: Pro-Val-Gly-Val-Val (SEQ ID NO. 55).
According to a particular embodiment, (W)w can be a dipeptide (i.e. w = 2) such
as Val-Cit, or the linker lacks an amino acid unit (w=0). When the linker lacks an amino
acid unit, preferably it lacks also a spacer unit.
According to a preferred embodiment, w = 0 (i.e. (W)w is a single bond) or w =
2 (i.e. (W)w is a dipeptide) and (W)wcan thus be selected from:
Ala-Ala),
and in particular is Val-Cit,
wherein
the asterisk indicates the point of attachment to the spacer unit if present, or to
the drug D; and
the wavy line indicates the point of attachment to L2.
Amino acid linker components can be designed and optimized in their selectivity
for enzymatic cleavage by a particular enzyme, for example, a tumor-associated
protease, cathepsin B, C and D, or a plasmin protease.
The amino acid unit of the linker can be enzymatically cleaved by an enzyme
including, but not limited to, a tumor-associated protease to liberate the drug.
The amino acid unit can be designed and optimized in its selectivity for
enzymatic cleavage by a particular tumor-associated protease. The suitable units are
those whose cleavage is catalyzed by the proteases, cathepsin B, C and D, and plasmin.
The spacer unit (Y), when present, links an amino acid unit if present, or the
stretcher unit if present, or otherwise the antibody to the drug. Spacer units are of two
general types: self-immolative and non self-immolative. A non self-immolative spacer
unit is one in which part or all of the spacer unit remains bound to the drug after
enzymatic cleavage of an amino acid unit from the antibody-drug conjugate. Examples
of a non self-immolative spacer unit include, but are not limited to a (glycine-glycine)
spacer unit and a glycine spacer unit. To liberate the drug, an independent hydrolysis
reaction should take place within the target cell to cleave the glycine-drug unit bond.
In a particular embodiment, a non self-immolative the spacer unit (Y) is Gly.
Alternatively, an antibody-drug conjugate containing a self-immolative spacer
unit can release the drug without the need for a separate hydrolysis step. In these
embodiments, (Y) is a residue of p-aminobenzyl alcohol (PAB) unit that is linked to
(W)w via the nitrogen atom of the PAB group, and connected directly to the drug via a
ester, carbonate, carbamate or ether group.
Other examples of self-immolative spacers include, but are not limited to,
aromatic compounds that are electronically equivalent to the PAB group such as
residues of 2-aminoimidazol-5-methanol derivatives and ortho or paraaminobenzylacetals.
Spacers can be used that undergo facile cyclization upon amide
bond hydrolysis, such as substituted and unsubstituted 4-aminobutyric acid amides,
appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems and 2-
aminophenylpropionic acid amides.
In an alternate embodiment, the spacer unit is a branched
bis(hydroxymethyl)styrene (BHMS) unit, which can be used to incorporate additional
drugs.
In a particular embodiment, the spacer unit (Y) is PAB-carbonyl with PAB being
(the oxygen of the PAB unit being linked to the carbonyl),
1 or the linker lacks a spacer unit (y=0).
In a particular embodiment, the linker has the following formula (III):
(III)
wherein
L2 is (C4 -Cio)cycloalkyl-carbonyl, (C2-C )alkyl or (C2-C )alkyl-carbonyl (the
carbonyl of these moieties, when present, being linked to (W)w),
W represents an amino acid unit, with w representing an integer comprised
between 0 and 5,
Y is PAB-carbonyl, with PAB being (the oxygen of
the PAB unit being linked to the carbonyl), and y is 0 or 1 (preferably y is 0 when w is 0
and y is 0 or 1when w is comprised between 1 and 5),
the asterisk indicates the point of attachment to the drug D, and
the wavy line indicates the point of attachment to the antibody Ab.
Advantageously, L2 is (C2-C )alkyl-carbonyl such as a pentyl-carbonyl of the
following formula:
wherein
the asterisk indicates the point of attachment to (W)w; and
the wavy line indicates the point of attachment to the nitrogen atom of the
maleimide moiety.
Accordin to a preferred embodiment, the linker L is selected from:
wherein the asterisk indicates the point of attachment to the drug D, and the
wavy line indicates the point of attachment to the antibody Ab.
IV - The Antibody-drug-conjugate (ADC)
In a preferred embodiment, the antibody-drug conjugate of the invention may be
prepared by any method known by the person skilled in the art such as, without
limitation, i) reaction of a nucleophilic group of the antibody with a bivalent linker
reagent followed by reaction with a nucleophilic group of the drug or ii) reaction of a
nucleophilic group of the drug with a bivalent linker reagent followed by reaction with a
nucleophilic group of the antibody.
Nucleophilic groups on antibody include, without limitation, N-terminal amine
groups, side chain amine groups (e.g. lysine), side chain thiol groups, and sugar
hydroxyl or amino groups when the antibody is glycosylated.
Nucleophilic groups on the drug include, without limitation, amine, thiol, and
hydroxyl groups, and preferably amine groups.
Amine, thiol, and hydroxyl groups are nucleophilic and capable of reacting to
form covalent bonds with electrophilic groups on linker moieties and linker reagents
including, without limitation, active esters such as NHS esters, HOBt esters,
haloformates, and acid halides; alkyl and benzyl halides such as haloacetamides;
aldehydes; ketones; carboxyl; and maleimide groups. The antibody may have reducible
interchain disulfides, i.e. cysteine bridges. The antibody may be made reactive for
conjugation with linker reagents by treatment with a reducing agent such as DTT
(dithiothreitol). Each cysteine bridge will thus form, theoretically, two reactive thiol
nucleophiles. Additional nucleophilic groups can be introduced into the antibody
through any reaction known by the person skilled in the art. As non limitative example,
reactive thiol groups may be introduced into the antibody by introducing one or more
cysteine residues.
Antibody-drug conjugates may also be produced by modification of the antibody
to introduce electrophilic moieties, which can react with nucleophilic substituents on the
linker reagent. The sugars of glycosylated antibody may be oxidized to form aldehyde
or ketone groups which may react with the amine group of linker reagents or drug. The
resulting imine Schiff base groups may form a stable linkage, or may be reduced to
form stable amine linkages. In one embodiment, reaction of the carbohydrate portion of
a glycosylated antibody with either galactose oxidase or sodium meta-periodate may
yield carbonyl (aldehyde and ketone) groups in the protein that can react with
appropriate groups on the drug. In another embodiment, proteins containing N-terminal
serine or threonine residues can react with sodium meta-periodate, resulting in
production of an aldehyde in place of the first amino acid.
In a preferred embodiment, the antibody-drug conjugate of the invention is
prepared by preparation of the drug-linker moiety followed by coupling between a
nucleophilic group of the antibody (for ex. the SH group of a cysteine moiety) and an
electrophilic group of the drug-linker moiety (for ex. a maleimide).
1. Drug-Linker
The Drug-Linker moiety can be prepared by coupling:
- the linker with the drug,
- a part of the linker with the drug before completing the synthesis of the linker,
- the linker with a part or a precursor of the drug before completing the synthesis of
the drug, or
- a part of the linker with a part or a precursor of the drug before completing the
synthesis of the linker and the drug.
The coupling reactions are well known reactions for the one skilled in the art
between a nucleopilic group and an electrophilic group.
The nucleophilic group can be in particular an amine, thiol or hydroxyl group. In
a preferred embodiment it is a primary or secondary amine group.
The electrophilic group can be a carboxylic acid group (COOH) optionally in an
activated form or an activated carbonate ester moiety.
By "activated form" of a carboxylic acid is meant a carboxylic acid in which the
OH moiety of the COOH function has been replaced with an activated leaving group
(LG) enabling coupling of the activated carboxylic acid group with an amino group in
order to form an amide bond and release the compound LG-H. Activated forms may be
activated esters, activated amides, anhydrides or acyl halides such as acyl chlorides.
Activated esters include derivatives formed by reaction of the carboxylic acid group
with N-hydroxybenzotriazole or N-hydroxysuccinimide.
By "activated carbonate ester" is meant a carbonate ester comprising
a -OC(0 )OR moiety in which OR represents a good leaving group enabling coupling of
the activated carbonate ester with an amino group in order to form a carbamate moiety
and release the compound ROH. The R group of the activated carbonate ester includes,
without limitation, the p-nitro-phenyl, pentafluorophenyl, 2,5-dioxo-2,5-dihydro-l Hpyrrol-
l-yl and benzyl groups, preferably the p-nitro-phenyl and pentafluorophenyl
groups.
When the linker has the following formula (III):
(III)
the Drug-Linker moiety has the following formula (IV):
(IV)
and the last step of the synthesis of the Drug-Linker moiety is generally the coupling
between a compound of the following formula (V):
(V)
where L2 is as defined previously and LG represents a leaving group notably a halide
such as a chloride or a group derived from N-hydroxysuccinimide,
and a compound of the following formula (VI):
H (W)w -(Y) —D
(VI).
When y = 1 and Y = PAB-carbonyl, the compound of formula (VI) can be
prepared by the coupling between the drug (DH) and a compound of the following
formula (VII), preferably a protected form thereof:
G-(W)W-PAB—CO—OR
(VII)
where W and w are as defined previously and R is as defined in the definition of the
"activated carbonate ester", and G is H or a protecting group.
When the compound of formula (VII) is in a protected form, final step of
deprotection is necessary.
When y = 0, the compound (VI) has the formula H-(W)W-D, wherein (W)w and
preferably D are composed of amino acid units. Consequently, the compound (VI) can
be prepared in this case by a conventional peptide synthesis method well known to the
one skilled in the art.
2. Ab-Linker-Drug
A preferred embodiment according to the invention consists of a coupling
between a cysteine present on the antibody and an electrophilic group of the Drug-
Linker moiety, preferably with a maleimide moiety present on the Drug-Linker moiety.
The maleimide-cysteine coupling can be performed by methods well known to
the person skilled in the art.
Generally, antibodies do not contain many, if any, free and reactive cysteine
thiol groups which can be linked to a drug moiety. Most cysteine thiol residues in
antibodies exist as disulfide bridges and must be reduced with a reducing agent such as
dithiothreitol (DTT) or TCEP, under partial or total reducing conditions. The loading
(drug/antibody ratio) of an ADC may be controlled in several different manners,
including: (i) limiting the molar excess of drug-linker intermediate (D-L) or linker
reagent relative to antibody, (ii) limiting the conjugation reaction time or temperature,
and (iii) partial or limited reducing conditions for cysteine thiol modification.
The disulfide bond structure of human IgGs is now well established (reviewed in
Liu and May, mAbs 4 (2012): 17-23). There are in fact many similarities and some
differences with regard to the disulfide bond structures of the 4 human IgG subclasses,
namely IgGl, IgG2, IgG3 and IgG4. All IgG subclasses contain invariably 12 intrachain
disulfide bridges and the differences reside in their inter-chain disulfide bonds
formed between heavy and light chains. Each intra-chain disulfide bond is associated
with an individual IgG domain, i.e. variable (VL and VH) and constant (CL, CHI, CH2
and CH3) domains. The 2 heavy chains are linked in their hinge region by a variable
number of disulfide bridges: 2 for IgGl and IgG4, 4 for IgG2 and 11 for IgG3. The
heavy and light chains of the IgGl are connected by a disulfide bond between the last
cysteine residue of the light chain and the fifth residue of the heavy chain, whereas for
the other subclasses, IgG2, IgG3 and IgG4, the light chain is linked to the heavy chain
by a disulfide bond between the last cysteine residue of the light chain and the third
cysteine residue of the heavy chain, which is located at the interface of VH and CHI
domains. Disulfide bond structures other than these classical structures have been
described for IgG2 and IgG4 (reviewed in Liu and May, mAbs 4 (2012): 17-23). Interchain
disulfide bonds are highly solvent exposed and are consequently much more
reactive than the intra-chain disulfide bonds, which are buried in anti-parallel beta-sheet
structures within each domain and are not solvent exposed. For these reasons, whatever
the antibody isotype, coupling will take place on inter-chain exposed cysteine residues
after mild reduction. Each inter-chain disulfide bridge can thus form, theoretically, two
sites of conjugation.
Additional nucleophilic groups can be introduced into antibodies through the
reaction of lysines with 2-iminothiolane (Traut's reagent) resulting in the conversion of
an amine into a thiol. Reactive thiol groups may also be introduced into the antibody
(or fragment thereof) by engineering one, two, three, four, or more cysteine residues
(e.g., preparing mutant antibodies comprising one or more non-native cysteine amino
acid residues). US 7521541 teaches engineering antibodies by introduction of reactive
cysteine amino acids.
Cysteine amino acids may be engineered at reactive sites in an antibody and
which do not form intrachain or intermolecular disulfide linkages (Junutula, et al,
2008b Nature Biotech., 26(8):925-932; Dornan et al (2009) Blood 114(13):2721-2729;
US 7521541; US 7723485; WO2009/052249). The engineered cysteine thiols may
react with linker reagents or the drug-linker reagents of the present invention which
have thiol-reactive, electrophilic groups such as maleimide or alpha-halo amides to form
ADC with cysteine engineered antibodies and the drug moieties. The location of the
drug moiety can thus be designed, controlled, and known. The drug loading can be
controlled since the engineered cysteine thiol groups typically react with thiol-reactive
linker reagents or drug-linker reagents in high yield. Engineering an IgG antibody to
introduce a cysteine amino acid by substitution at a single site on the heavy or light
chain gives two new cysteines on the symmetrical antibody. A drug loading near 2 can
be achieved with near homogeneity of the conjugation product ADC.
Where more than one nucleophilic or electrophilic group of the antibody reacts
with a drug-linker intermediate, or linker reagent followed by drug moiety reagent, then
the resulting product is a mixture of ADC compounds with a distribution of drug
moieties attached to an antibody, e.g. 1, 2, 3, etc. Liquid chromatography methods such
as polymeric reverse phase (PLRP) and hydrophobic interaction (HIC) may separate
compounds in the mixture by drug loading value. Preparations of ADC with a single
drug loading value (p) may be isolated, however, these single loading value ADCs may
still be heterogeneous mixtures because the drug moieties may be attached, via the
linker, at different sites on the antibody.
For some antibody-drug conjugates, drug ratio may be limited by the number of
attachment sites on the antibody. High drug loading, e.g. drug ratio >5, may cause
aggregation, insolubility, toxicity, or loss of cellular permeability of certain antibodydrug
conjugates. Typically, less drug moieties than the theoretical maximum are
conjugated to an antibody during a conjugation reaction.
The drug loading also referred as the Drug-Antibody ratio (DA ) is the average
number of drugs per cell binding agent.
In the case of antibody IgGl and IgG4 isotypes, where the drugs are bound to
cysteines after partial antibody reduction, drug loading may range from 1 to 8 drugs (D)
per antibody, i.e. where 1, 2, 3, 4, 5, 6, 7, and 8 drug moieties are covalently attached to
the antibody.
In the case of an antibody IgG2 isotype, where the drugs are bound to cysteines
after partial antibody reduction, drug loading may range from 1 to 12 drugs (D) per
antibody, i.e. where 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 drug moieties are covalently
attached to the antibody.
Compositions of ADC include collections of cell binding agents, e.g. antibodies,
conjugated with a range of drugs, from 1 to 8 or 1 to 12.
The average number of drugs per antibody in preparations of ADC from
conjugation reactions may be characterized by conventional means such as UV, reverse
phase HPLC, HIC, mass spectrometry, ELISA assay, and electrophoresis.
As non limitative embodiment, it is presented herein the conjugation with the
antibody c208F2. In this case, the drug is coupled to at least one cysteine selected from
i) for the light chain of sequence SEQ ID No. 28, the residue Cys. in position 214 and ii)
for the heavy chain of sequence SEQ ID No. 23, the residues Cys. in position 223, 229
and 232.
As non limitative embodiment, it is presented herein the conjugation with the
antibody c208F2. In this case, the drug is coupled to two, three or four, cysteines
selected from i) for the light chain of sequence SEQ ID No. 28, the residue Cys. in
position 214 and ii) for the heavy chain of sequence SEQ ID No. 23, the residues Cys.
in position 223, 229 and 232
As non limitative embodiment, it is presented herein the conjugation with the
antibody hz208F2 (ar. 1). In this case, the drug is coupled to at least one cysteine
selected from i) for the light chain of sequence SEQ ID No. 39, the residue Cys. in
position 214 and ii) for the heavy chain of sequence SEQ ID No. 37, the residues Cys.
in position 223, 229 and 232.
As non limitative embodiment, it is presented herein the conjugation with the
antibody hz208F2 (var. 3). In this case, the drug is coupled to two, three or four,
cysteines selected from i) for the light chain of sequence SEQ ID No. 40, the residue
Cys. in position 214 and ii) for the heavy chain of sequence SEQ ID No. 38, the
residues Cys. in position 223, 229 and 232
An alternative consists of lysine coupling. An antibody may contain, for
example, many lysine residues that do not react with the drug-linker intermediate (D-L)
or linker reagent. Only the most reactive lysine groups may react with an aminereactive
linker reagent. Also, only the most reactive cysteine thiol groups may react
with a thiol-reactive linker reagent.
Where the compounds of the invention are bound to lysines, drug loading may
range from 1 to 80 drugs (D) per cell antibody, although an upper limit of 40, 20, 10 or
8 may be preferred. Compositions of ADC include collections of cell binding agents,
e.g. antibodies, conjugated with a range of drugs, from 1 to 80, 1 to 40, 1 to 20, 1 to 10
or 1 to 8.
The ADC of formula (I) according to the invention can be in the form of a
pharmaceutically acceptable salt.
In the present invention by "pharmaceutically acceptable" is meant that which
can be used in the preparation of a pharmaceutical composition which is generally, safe
non-toxic and neither biologically nor otherwise undesirable, and which is acceptable
for veterinary use as well as for human pharmaceutical use.
By "pharmaceutically acceptable salt" of a compound is meant a salt which is
pharmaceutically acceptable as defined herein and which has the desired
pharmacological activity of the parent compound.
Pharmaceutically acceptable salts notably comprise:
(1) the addition salts of a pharmaceutically acceptable acid formed with
pharmaceutically acceptable inorganic acids such as hydrochloric, hydrobromic,
phosphoric, sulfuric and similar acids; or formed with pharmaceutically acceptable
organic acids such as acetic, trifluoroacetic, propionic, succinic, fumaric, malic, tartaric,
citric, ascorbic, maleic, glutamic, benzoic, salicylic, toluenesulfonic, methanesulfonic,
stearic, lactic and similar acids; and
(2) the addition salts of a pharmaceutically acceptable base formed when an acid
proton present in the parent compound is either replaced by a metallic ion e.g. an
alkaline metal ion, an alkaline-earth metal ion or an aluminium ion; or coordinated with
a pharmaceutically acceptable organic base such as lysine, arginine and similar; or with
a pharmaceutically acceptable inorganic base such as sodium hydroxide, potash,
calcium hydroxide and similar.
These salts can be prepared from the compounds of the invention containing a
base or acid function, and the corresponding acids or bases using conventional chemical
methods.
V - Treatment
Finally, the invention relates to an ADC as above described for use as a drug, in
particular in the treatment of cancer.
A further subject of the present invention is a formal (I) compound such as
defined above for use as medicinal product, in particular for the treatment of cancer.
The present invention also concerns the use of a formula (I) compound such as
defined above for producing a medicinal product, particularly intended for the treatment
of cancer.
The present invention also concerns a method for treating cancer comprising the
administration to a person in need thereof of an effective mount of a formula (I)
compound such as defined above.
Cancers can be preferably selected through IGF-lR-related cancers including
tumoral cells expressing or over-expressing whole or part of the protein IGF-IR at their
surface.
More particularly, said cancers are breast cancer, colon cancer, esophageal
carcinoma, hepatocellular cancer, gastric cancer, glioma, lung cancer, melanoma,
osteosarcoma, ovarian cancer, prostate cancer, rhabdomyosarcoma, renal cancer,
thyroid cancer, uterine endometrial cancer, schwannoma, neuroblastoma, oral squamous
cancer, mesothelioma, leiomyosarcoma and any drug resistance phenomena or cancers.
For the avoidance of doubt, by drug resistance IGF-lR-expressing cancers, it
must be understood not only resistant cancers which initially express IGF-IR but also
cancers which initially do not express or overexpress IGF-IR but which express IGF-IR
once they have become resistant to a previous treatment.
Another object of the invention is a pharmaceutical composition comprising the
ADC as described in the specification.
More particularly, the invention relates to a pharmaceutical composition
comprising the ADC of the invention with at least an excipient and/or a pharmaceutical
acceptable vehicle.
In the present description, the expression "pharmaceutically acceptable vehicle"
or "excipient" is intended to indicate a compound or a combination of compounds
entering into a pharmaceutical composition not provoking secondary reactions and
which allows, for example, facilitation of the administration of the active compound(s),
an increase in its lifespan and/or in its efficacy in the body, an increase in its solubility
in solution or else an improvement in its conservation. These pharmaceutically
acceptable vehicles and excipients are well known and will be adapted by the person
skilled in the art as a function of the nature and of the mode of administration of the
active compound(s) chosen.
The active ingredient can be administered in unit forms of administration, in a
mixture with conventional pharmaceutical carriers, to animals or to human beings.
Suitable unit forms of administration comprise forms via oral route and forms for
administration via parenteral route (subcutaneous, intradermal, intramuscular or
intravenous).
As solid compositions, for oral administration, use can be made of tablets, pills,
powders (hard or soft gelatine capsules) or granules. In these compositions, the active
ingredient of the invention is mixed with one or more inert diluents such as starch,
cellulose, sucrose, lactose or silica, in a stream of argon. These compositions may also
comprise substances other than diluents, for example one or more lubricants such as
magnesium stearate or talc, a colouring agent, a coating (coated tablets) or a varnish.
The sterile compositions for parenteral administration may preferably be
aqueous or non-aqueous solutions, suspensions or emulsions. As solvent or vehicle, use
can be made of water, propylene glycol, a polyethylene glycol, vegetable oils, in
particular olive oil, injectable organic esters e.g. ethyl oleate or other suitable organic
solvents. These compositions may also contain adjuvants, in particular wetting, isotonic,
emulsifying, dispersing and stabilising agents. Sterilisation can be performed in several
manners, for example by sanitising filtration, by incorporating sterilising agents into the
composition, by radiation or by heating. They can also be prepared in the form of solid
sterile compositions which can be dissolved at the time of use in sterile water or any
other injectable sterile medium.
Preferably, these ADCs will be administered by the systemic route, in particular
by the intravenous route, by the intramuscular, intradermal, intraperitoneal or
subcutaneous route, or by the oral route. In a more preferred manner, the composition
comprising the ADCs according to the invention will be administered several times, in a
sequential manner.
The invention concerns thus also a kit comprising at least i) an antibody-drugconjugate
according to the invention and/or a pharmaceutical composition according to
the invention and ii) a syringe or vial or ampoule in which the said antibody-drugconjugate
and/or pharmaceutical composition is disposed.
Their modes of administration, dosages and optimum pharmaceutical forms can
be determined according to the criteria generally taken into account in the establishment
of a treatment adapted to a patient such as, for example, the age or the body weight of
the patient, the seriousness of his/her general condition, the tolerance to the treatment
and the secondary effects noted.

CLAIMS
1. An antibody-drug-conjugate of the following formula (I):
Ab-(L-D)
(I)
or a pharmaceutically acceptable salt thereof,
wherein
Ab is an antibody, or an antigen binding fragment thereof, capable of binding to
the human IGF-IR selected from i) an antibody which comprises the three heavy chain
CDRs of sequence SEQ ID No. 1, 2 and 3 and the three light chain CDRs of sequence
SEQ ID No. 4, 5 and 6; or ii) an antibody which competes for binding to IGF-IR with
the antibody of i); or iii) an antibody which binds to the same epitope of IGF-IR as the
antibody of i);
L is a linker;
D is a drug moiety of the following formula (II):
(II)
wherein:
R2 is COOH, COOCH3 or thiazolyl;
R3is H or (Ci-C 6)alkyl;
R is H or (Ci-C 6)alkyl;
m is an integer comprised between 1 and 8;
the wavy line indicates the point of attachment to L; and
n is 1 to 12.
2. The antibody-drug-conjugate of claim 1, wherein Ab is selected from:
a) an antibody comprising the three heavy chain CDRs of sequence SEQ ID No.
7, 2 and 3 and the three light chain CDRs of sequence SEQ ID No. 9, 5 and 11;
b) an antibody comprising the three heavy chain CDRs of sequence SEQ ID No.
7, 2 and 3 and the three light chain CDRs of sequence SEQ ID No. 10, 5 and 11;
c) an antibody comprising the three heavy chain CDRs of sequence SEQ ID No.
7, 2 and 3 and the three light chain CDRs of sequence SEQ ID No. 9, 5 and 12; and
d) an antibody comprising the three heavy chain CDRs of sequence SEQ ID No.
8, 2 and 3 and the three light chain CDRs of sequence SEQ ID No. 9, 5 and 11.
3. The antibody-drug-conjugate of claim 1 or 2, wherein Ab is selected from:
a) an antibody comprising a heavy chain variable domain of sequence
SEQ ID No. 13 and the three light chain CDRs of sequence SEQ ID No. 9, 5 and 11;
b) an antibody comprising a heavy chain variable domain of sequence
SEQ ID No. 14 and the three light chain CDRs of sequence SEQ ID No. 10, 5 and 11;
c) an antibody comprising a heavy chain variable domain of sequence
SEQ ID No. 15 and the three light chain CDRs of sequence SEQ ID No. 9, 5 and 12;
d) an antibody comprising a heavy chain variable domain of sequence
SEQ ID No. 16 and the three light chain CDRs of sequence SEQ ID No. 9, 5 and 11;
and
e) an antibody comprising a heavy chain variable domain of sequence
SEQ ID No. 17 and the three light chain CDRs of sequence SEQ ID No. 9, 5 and 12.
4. The antibody-drug-conjugate of claim 1 or 2, wherein Ab is selected from:
a) an antibody comprising a light chain variable domain of sequence
SEQ ID No. 18 and the three heavy chain CDRs of sequence SEQ ID No. 7, 2 and 3;
b) an antibody comprising a light chain variable domain of sequence
SEQ ID No. 19 and the three heavy chain CDRs of sequence SEQ ID No. 7, 2 and 3;
c) an antibody comprising a light chain variable domain of sequence
SEQ ID No. 20 and the three heavy chain CDRs of sequence SEQ ID No. 7, 2 and 3;
d) an antibody comprising a light chain variable domain of sequence
SEQ ID No. 2 1 and the three heavy chain CDRs of sequence SEQ ID No. 8, 2 and 3;
and
e) an antibody comprising a light chain variable domain of sequence
SEQ ID No. 22 and the three heavy chain CDRs of sequence SEQ ID No. 7, 2 and 3.
5. The antibody-drug-conjugate of claim 1, wherein Ab is selected from i) the
antibodies 208F2, 212A1 1, 214F8, 219D6 and 213B10, ii) the antibodies which
compete for binding to IGF-1R with the antibodies of i); and iii) the antibodies which
bind to the same epitope of IGF-1R as the antibodies of i).
6. The antibody-drug-conjugate of claim 1, wherein Ab comprises:
a) a heavy chain variable domain (VH) of sequence SEQ ID No. 33 wherein said
sequence SEQ ID No. 33 comprises at least 1 back-mutation selected from the residues
20, 34, 35, 38, 48, 50, 59, 61, 62, 70, 72, 74, 76, 77, 79, 82 and 95; and
b) a light chain variable domain (VL) of sequence SEQ ID No. 35, wherein said
sequence SEQ ID No. 35 comprises at least 1 back-mutation selected from the residues
22, 53, 55, 65, 71, 72, 77 or 87.
7. The antibody-drug-conjugate of claim 1, wherein Ab is selected from:
a) an antibody comprising a heavy chain variable domain of sequence selected
from SEQ ID Nos. 56, 62, 64, 66, 68, 70, 72, 74, 76, 78 and 80 or any sequence with at
least 80% identity with SEQ ID No.56, 62, 64, 66, 68, 70, 72, 74, 76, 78 or80; and the
three light chain CDRs of sequences SEQ ID Nos. 9, 5 and 11;
b) an antibody comprising a light chain variable domain of sequence selected
from SEQ ID Nos. 57 or 60 or any sequence with at least 80% identity with SEQ ID
Nos. 57 or 60; and the three heavy chain CDRs of sequences SEQ ID Nos. 7, 2 and 3;
and
c) an antibody comprising a heavy chain variable domain of sequence selected
from SEQ ID Nos. 56, 62, 64, 66, 68, 70, 72, 74, 76, 78 and 80 or any sequence with at
least 80% identity with SEQ ID Nos.56, 62, 64, 66, 68, 70, 72, 74, 76, 78 or 80; and a
light chain variable domain of sequence selected from SEQ ID Nos. 57 or 60 or any
sequence with at least 80%> identity with SEQ ID Nos. 57 or 60.
8. The antibody-drug-conjugate of claim 1, wherein Ab comprises:
a) a heavy chain of sequence selected from SEQ ID Nos. 58, 63, 65, 67, 69, 71, 73, 75,
77, 79 and 8 1 or any sequence with at least 80%> identity with SEQ ID Nos. 58, 63, 65,
67, 69, 71, 73, 75, 77, 79 or 81; and
b) a light chain of sequence selected from SEQ ID Nos. 59 and 6 1 or any sequence with
at least 80% identity with SEQ ID Nos. 59 or 61.
9. The antibody-drug-conjugate of any of the preceding claims, wherein L is a
linker of the following formula (III):
(III)
wherein
L2 is (C4 -Cio)cycloalkyl-carbonyl, (C2-C )alkyl, (C2-C )alkyl-carbonyl,
W is an amino acid unit; w is an integer comprised between 0 and 5;
Y is PAB-carbonyl with PAB being ; y is 0 or 1;
the asterisk indicates the point of attachment to D; and
the wavy line indicates the point of attachment to Ab.
10. The antibody-drug-conjugate of claim 9, wherein L2 is of the following
formula:
wherein
the asterisk indicates the point of attachment to (W)w; and
the wavy line indicates the point of attachment to the nitrogen atom of the
maleimide moiety.
11. The antibody-drug-conjugate of claim 9, wherein (W)w is selected from:
a sin le bond,
wherein
the asterisk indicates the point of attachment to (Y) ; and
the wavy line indicates the point of attachment to L2.
12. The antibody-drug-conjugate of claim 9, wherein w = 0; or w = 2 and (W)
is selected from:
nd
wherein
the asterisk indicates the point of attachment to (Y)y; and
the wavy line indicates the point of attachment to L2.
13. The antibody-drug-conjugate of any of the preceding claims, wherein (L-D)
is selected from:
(E-12)
200
G-13)
(F-61)
14. An antibody-drug-conjugate according to claim 1 having the formula
selected from:
(Ab-G-12)
(Ab-G-13)
(Ab-F-61)
-F-62)
(Ab-F-63)
and the pharmaceutically acceptable salts thereof,
wherein Ab is selected in the group consisting of i) the antibodies 208F2,
212A1 1, 214F8, 219D6 and 213B10, and ii) the antibodies which compete for binding
to IGF-1R with the antibodies of i); and iii) the antibodies which bind to the same
epitope of IGF-1R as the antibodies of i).
15. The antibody-drug-conjugate of claim 1, wherein n is 2.
16. The antibody-drug-conjugate of claim 1, wherein n is 4.
17. The antibody-drug-conjugate of any of the preceding claims for use as a
medicament.
18. A composition comprising at least one antibody-drug-conjugate of any of the
preceding claims.
19. The composition of claim 18 further comprising a pharmaceutically
acceptable vehicle.
20. The composition of claim 18 or 19 for use in the treatment of an IGF-1Rexpressing
cancer.
21. The composition of claim 20, wherein said IGF-lR-expressing cancer is
a cancer chosen from breast, colon, esophageal carcinoma, hepatocellular, gastric,
glioma, lung, melanoma, osteosarcoma, ovarian, prostate, rhabdomyosarcoma, renal,
thyroid, uterine endometrial cancer, mesothelioma, oral squamous carcinoma and any
drug resistant cancer.
22. A method for the treatment of an IGF-lR-expressing cancer in a subject in
need thereof, comprising administering to the subject an effective amount of at least one
antibody-drug-conjugate of any of claims 1 to 16 or of a composition of claim 18 or 19.